Monoclonal antibodies against amyloid beta protein and uses thereof

ABSTRACT

The subject invention relates to monoclonal antibodies (e.g., 8F5 and 8C5) that may be used, for example, in the prevention, treatment and diagnosis of Alzheimer&#39;s Disease or other neurodegenerative disorders.

BACKGROUND OF THE INVENTION

1. Technical Field

The subject invention relates to monoclonal antibodies (e.g., 8F5 andBC5) that may be used, for example, in the prevention, treatment anddiagnosis of Alzheimer's Disease or other neurodegenerative disorders.

2. Background Information

Alzheimer's Disease (AD) is a neurodegenerative disorder characterizedby a progressive loss of cognitive abilities and by characteristicneuropathological features comprising amyloid deposits, neurofibrillarytangles and neuronal loss in several regions of the brain see Hardy andSelkoe (Science 297, 353 (2002); Mattson (Nature 431, 7004 (2004). Theprincipal constituents of amyloid deposits are amyloid beta-peptides(Aβ), with the 42 amino acid-long type (Aβ1-42) being the mostprominent,

In particular, amyloid β(1-42) protein is a polypeptide having 42 aminoacids which is derived from the amyloid precursor protein (APP) byproteolytic processing. This also includes, in addition to humanvariants, isoforms of the amyloid β(1-42) protein present in organismsother than humans, in particular, other mammals, especially rats. Thisprotein, which tends to polymerize in an aqueous environment, may bepresent in very different molecular forms.

A simple correlation of the deposition of insoluble protein with theoccurrence or progression of dementia disorders such as, for example,Alzheimer's disease, has proved to be unconvincing (Terry et al., Ann.Neurol. 30, 572-580 (1991); Dickson et al., Neurobiol. Aging 16, 285-298(1995)). In contrast, the loss of synapses and cognitive perceptionseems to correlate better with soluble forms of Aβ(1-42) (Lue et al.,Am. J. Pathol. 155, 853-862 (1999); McLean et al., Ann. Neurol. 46,860-866 (1999)).

Although polyclonal and monoclonal antibodies have been raised in thepast against Aβ(1-42), none have proven to produce the desiredtherapeutic effect without also causing serious side effects in animalsand/or humans. For example, passive immunization results frompreclinical studies in very old APP23 mice which received a N-terminaldirected anti-A62(1-42) antibody once weekly for 5 months indicatetherapeutically relevant side effect. In particular, these mice showedan increase in number and severity of microhemorrhages compared tosaline-treated mice (Pfeifer et al., Science 2002 298:1379), A similarincrease in hemorrhage was recently also described for very old (>24months) Tg2576 and PDAPP mice (Wilcock et al., J Neuroscience 2003, 23:3745-51; Racke et al., J Neuroscience 2005, 25:629-636). In bothstrains, injection of anti-Aβ(1-42) resulted in a significant increaseof microhemorrhages, Thus, a tremendous therapeutic need exists for thedevelopment of biologics that prevent or slow down the progression ofthe disease without inducing negative and potentially lethal effects onthe human body, Such need is particularly evident in view of theincreasing longevity of the general population and, with this increase,an associated rise in the number of patents annually diagnosed with

Alzheimer's Disease. Further, such antibodies will allow for properdiagnosis of Alzheimer's Disease in a patient experiencing symptomsthereof, a diagnosis which can only be confirmed upon autopsy at thepresent time. Additionally, the antibodies will allow for theelucidation of the biological properties of the proteins and otherbiological factors responsible for this debilitating disease.

All patents and publications referred to herein are hereby incorporatedin their entirety by reference.

SUMMARY OF THE INVENTION

The present invention includes an isolated antibody that binds withgreater specificity to an amyloid beta (Aβ) protein globulomer than toan amyloid beta protein monomer. Thus, preferential binding is observed.The antibody may be, for example, a monoclonal antibody such as 8F5 or805. The ratio of binding specificity to the globulomer versus themonomer is at least 1.4. In particular, the ratio is preferably at leastabout 1.4 to at least about 16.9. (A ratio of 1.0-17,5 including theendpoints) is also considered to fall within the scope of the presentinvention as well as decimal percentages thereof. For example, 1.1, 1.2,1.3, 2.0, 2.1, 2.2 . . . , 17.1, 17.2, 17.3, 17.4, 17.5 as well as allfull integers in between and percentages thereof are considered to fallwithin the scope of the present invention.) The amyloid beta proteinmonomer may be, for example, Aβ(1-42) monomer or Aβ(1-40) monomer.

Further, the present invention also encompasses a monoclonal antibody(referred to herein as “8F5”) produced by a hybridoma having AmericanType Culture Collection designation number PTA-7238 as well as thehybridoma that produces this monoclonal antibody (i.e., 8F5). Also, thepresent invention includes a monoclonal antibody (referred to herein as“805”) produced by a hybridoma having American Type Culture Collectiondesignation number PTA-7407 as well as the hybridoma that produces thismonoclonal antibody (i.e., 805). Additionally, the present inventionincludes a monoclonal antibody comprising a variable heavy chain encodedby SEQ ID NO:1. This antibody may be murine, human or humanized.

Further, the present invention includes a monoclonal antibody comprisinga variable light chain encoded by SEQ ID NO:2. This antibody may also bemurine, human or humanized. The antibody may further comprise a variablelight heavy chain encoded by SEQ ID NO:1 and may be human or humanized.

Moreover, the present invention includes a monoclonal antibodycomprising SEQ ID NO:3. The antibody may be murine, human or humanized,

Further, the present invention encompasses a monoclonal antibodycomprising SEQ ID NO:4. This antibody may be murine, human or humanized.This antibody may further comprise SEQ ID NO:3 and may be murine, humanor humanized.

Additionally, the present invention includes a monoclonal antibodycomprising a variable heavy chain encoded by SEQ ID NO:11. This antibodymay be murine, human or humanized.

Further, the present invention includes a monoclonal antibody comprisinga variable light chain encoded by SEQ ID NO:12. This antibody may alsobe murine, human or humanized, The antibody may further comprise avariable heavy chain encoded by SEQ ID NO:11 and may be human orhumanized.

Moreover, the present invention includes a monoclonal antibodycomprising SEQ ID NO:19. The antibody may be murine, human or humanized.

Further, the present invention encompasses a monoclonal antibodycomprising SEQ ID NO:20. This antibody may be murine, human orhumanized, This antibody may further comprise SEQ ID NO:19 and may bemurine, human or humanized.

The present invention also includes an isolated antibody which bindswith greater specificity to an amyloid beta protein globulomer than toan amyloid beta protein fibril.

This antibody may be, for example, monoclonal and may be the monoclonalantibody produced by the hybridoma having American Type CultureCollection designation number PTA-7243 or the hybridoma having AmericanType Culture Collection PTA-7407, The hybridomas producing thesemonoclonal antibodies also fall within the scope of the presentinvention,

Further, the present invention includes an antibody in which at leastone of the complementarity determining regions (CDRs) of the variableheavy chain is selected from the group consisting of SEQ ID NO:5, SEQ IDNO:6 and SEQ ID NO:7,

Moreover, the present invention also includes an antibody in which atleast one of the CDRs of the variable light chain is selected from thegroup consisting of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:10, Thisantibody may further comprise at least one CDR of the variable heavychain selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 andSEQ ID NO:7.

The present invention also includes an antibody in which at least one ofthe CDRs of the variable heavy chain is selected from the groupconsisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

Further, the present invention also encompasses an antibody in which atleast one of the CDRs of the variable light chain is selected from thegroup consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:16. Thisantibody may further comprises at least one CDR of the variable heavychain selected from the group consisting of SEQ ID NO:13, SEQ ID NO andSEQ ID NO:15.

Additionally, the present invention encompasses a method of treating orpreventing Alzheimer's Disease in a patient in need of the treatment orprevention, This method comprises administering any one or more of theisolated antibodies described above to the patient in an amountsufficient to effect the treatment or prevention,

The isolated antibody may be administered, for example, via a routeselected from the group consisting of intramuscular administration,intravenous administration and subcutaneous administration.

The present invention also includes a method of diagnosing Alzheimer'sDisease in a patient suspected of having this disease. This methodcomprises the steps of: 1) isolating a biological sample from thepatient; 2) contacting the biological sample with at least one of theantibodies described above for a time and under conditions sufficientfor formation of antigen/antibody complexes; and 3) detecting presenceof the antigen/antibody complexes in said sample, presence of thecomplexes indicating a diagnosis of Alzheimer's Disease in the patient.The antigen may be, for example, a globulomer or a portion or fragmentthereof which has the same functional properties as the full globulomer(e.g., binding activity).

Further, the present invention includes another method of diagnosingAlzheimer's Disease in a patient suspected of having this disease. Thismethod comprises the steps of: 1) isolating a biological sample from thepatient; 2) contacting the biological sample with an antigen for a timeand under conditions sufficient for the formation of antibody/antigencomplexes; 3) adding a conjugate to the resulting antibody/antigencomplexes for a time and under conditions sufficient to allow theconjugate to bind to the bound antibody, wherein the conjugate comprisesone of the antibodies described above, attached to a signal generatingcompound capable of generating a detectable signal; and 4) detecting thepresence of an antibody which may be present in the biological sample,by detecting a signal generated by the 30 signal generating compound,the signal indicating a diagnosis of Alzheimer's Disease in the patient.The antigen may be a globulomer or a portion or fragment thereof havingthe same functional properties as the full globulomer (e.g., bindingactivity).

The present invention includes an additional method of diagnosingAlzheimer's Disease in a patient suspected of having Alzheimer'sDisease. This method comprises the steps of: 1) isolating a biologicalsample from the patient; 2) contacting the biological sample withanti-antibody, wherein the anti-antibody is specific for one of theantibodies described above, for a time and under conditions sufficientto allow for formation of anti-antibody/antibody complexes, thecomplexes containing antibody present in the biological sample; 2)adding a conjugate to the resulting anti-antibody/antibody complexes fora time and under conditions sufficient to allow the conjugate to bind tobound antibody, wherein the conjugate comprises an antigen, which bindsto a signal generating compound capable of generating a detectablesignal; and 3) detecting a signal generated by the signal generatingcompound, the signal indicating a diagnosis of Alzheimer's Disease inthe patient. Further, the present invention includes a compositioncomprising any one or more of the antibodies described above (e.g., 8F5and 8C5).

The present invention includes another method of preventing or treatingAlzheimer's Disease in a patient in need of such prevention ortreatment. This method comprises the step of administering thecomposition described directly above to the patient in an amountsufficient to effect the prevention or treatment.

Additionally, the present invention encompasses a vaccine comprising atleast one of the antibodies described above and a pharmaceuticallyacceptable adjuvant.

Moreover, the present invention includes a further method of preventingor treating Alzheimer's Disease in a patient in need of such preventionor treatment. This method comprises the step of administering thevaccine noted above to the patient in an amount sufficient to effect theprevention or treatment. Further, the present invention encompasses amethod of identifying compounds suitable for active immunization of apatient predicted to develop Alzheimer's Disease. This methodcomprises: 1) exposing one or more compounds of interest to one or moreof the antibodies described above for a time and under conditionssufficient for the one or more compounds to bind to the antibody orantibodies; 2) identifying those compounds which bind to the antibody orantibodies, the identified compounds to be used in active immunizationin a patient predicted to develop Alzheimer's Disease, Also, the presentinvention includes a kit comprising: a) at least one of the isolatedantibodies described above and b) a conjugate comprising an antibodyattached to a signal-generating compound, wherein the antibody of theconjugate is different from the isolated antibody. The kit may alsoinclude a package insert with instructions as to how the components ofthe kit are to be utilized.

The present invention also encompasses a kit comprising: a) ananti-antibody to one of the antibodies described above and b) aconjugate comprising an antigen attached to a signal-generatingcompound. The antigen may be a globulomer or a fragment or portionthereof having the same functional characteristics as the globulomer(e.g,, binding activity). Again, the kit may also include a packageinsert with instructions as to how the components of the kit are to beutilized,

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the selectivity of 8F5 for globulomers versusAβ(1-42) monomers, Aβ(1-40) and sAPP, Selectivity factors for BF5 can becalculated as ratios between EC50 values (versus Aβ(1-42) monomer inHFIP: 555.8/90.74=6.1; versus Aβ(1-42) monomer in NH₄OH:1007/90.74=11.1; versus Aβ(1-40) monomer: 667.8/90.74=7.4 versus sAPP:>100)

FIG. 2 illustrates SDS-PAGE analysis of fibril bound heavy and lightchain antibodies (lanes 4, 6, 8) and corresponding non-bound freefractions (lanes 3, 5, 7) in the supernatants.

FIG. 3 illustrates Aβ42 and Aβ40 content in CSF samples from patientswith Mild Cognitive Impairment (MCI, left) or Alzheimer's disease (AD,right). In both groups, it can be observed that 8F5 picks up a higherproportion of Aβ(1-42) and less or an equal amount of Aβ(1-40) ifcompared to a standard antibody 6E10 or compared to direct sampleanalysis with the same ELISAs.

FIG. 4 illustrates novel object recognition index as time spent withunknown versus familiar object in three groups of APP transgenic mice(i.e., 6G1, 8F5, PBS) and one group of non-transgenic litter mates (wildtype). The animals (number given below columns) were immunized withmonoclonal antibodies 6G1 or 8F5 or treated with vehicle (i.e.,phosphate-buffered saline; PBS, and wild type) by once weeklyintraperitoneal injection for three weeks. On the day of the lastinjection, a novel object recognition task was performed, The differencebetween PBS and wild type groups indicated a cognitive deficit of APPtransgenic mice in this paradigm. PBS-injected mice performed at chancelevel (i.e., not significantly different from 50) while all other miceshowed object recognition (t-test; stars). When the performance ofantibody-treated APP transgenic mice was compared with control groups, asignificant difference was found versus PBS-treated but not versuswild-type mice (ANOVA with post-hoc t-test; circles) indicating thatantibody treatment reversed the cognitive deficit in these APPtransgenic mice.

FIG. 5(A) illustrates the DNA sequence (SEQ ID NO:1) of the variableheavy chain encoding the monoclonal antibody referred to herein as“8F5”, and FIG. 5(B) illustrates the DNA sequence (SEQ ID NO:2) of thevariable light chain encoding the monoclonal antibody 8F5.(Complementarity determining regions (CDRs) are underlined in eachsequence; see also FIG. 6).

FIG. 6(A) illustrates the amino acid sequence (SEQ ID NO:3) of thevariable heavy chain of monoclonal antibody 8F5, and FIG. 6(B)illustrates the amino acid sequence (SEQ ID

NO:4) of the variable light chain of monoclonal antibody 8F5, One CDR ofthe variable heavy chain is represented by the amino acid sequence SYGMS(SEQ ID NO:5). Another CDR of the variable heavy chain is represented bythe amino acid sequence ASINSNGGSTYYPDSVKG (SEQ ID NO:6), and anotherCDR of the variable heavy chain is represented by the amino acidsequence SGDY (SEQ ID NO:7). One CDR of the variable light chain isrepresented by the amino acid sequence RSSQSLVYSNGDTYLH (SEQ ID NO:8).Another CDR of the variable light chain is represented by the amino acidsequence KVSNRFS (SEQ ID NO:9), and another CDR of the variable lightchain is represented by the amino acid sequence SQSTHVPWT (SEQ IDNO:10). All of the above-described CDRs are underlined in FIGS. 6(A) and6(B).

FIG. 7 shows the binding of antibodies, at different concentrations, totransversal sections of the neocortices of

Alzheimer's disease (AD) patients or old APP transgenic mice. Inparticular, FIG. 7(A) illustrates verification of amyloid deposits byCongo Red staining as plaques in brain tissue and as cerebral amyloidangiopathy (CAA) in brain vessels in the APP transgenic mouse lineTg2576 and in an AD patient (RZ55). FIG. 7(B) illustrates that thestaining of parenchymal deposits of Aβ (amyloid plaques) in an ADpatient (RZ16) occurs only with 6G1 and the commercially availableantibody 6E10 while 8F5 and 8C5 show considerably weaker staining. FIG.7(C) illustrates that the strong staining of parenchymal deposits of Aβ(amyloid plaques) in TG2576 mice occurs only with 6G1 and thecommercially available antibody 6E10 while 8F5 and 8C5 show considerablyweaker staining. FIGS. 7(D)-(G) illustrate the quantification of theanalysis of Aβ plaque staining in the histological images using imageanalysis. Optical density values (0%=no staining) were calculated fromthe greyscale values of plaques subtracted by greyscale values ofbackground tissue. (FIG. (D)=binding of 0.7 μg/ml antibody in Tg2576mice; FIG. (E)=binding of 0.07-0.7 μg/ml antibody in APP/L mice; FIG.(F)=binding of 0.7 g/ml antibody in an AD patient (RZ55); and FIG.(G)=binding of 0.07-0.7 μg/ml antibody in an AD patient (RZ16)) Thedifferences between staining of the commercially available antibodies6E10 (starts) and 4G8 (circles) and antibodies 6G1, 8C5 and 8F5 (oneasterisk/circle: p<0.05, two asterisks/circles: p<0.01, and threeasterisks/circles: p<0.001 versus control; post-hoc Bonferroni's t-testafter ANOVA with p<0.001) were statistically evaluated (FIG. (D) andFIG. (E)). In FIGS. (E) and (G), the antibodies 8C5 and 8F5 alwaysshowed significantly less staining than the commercially availableantibodies 6E10 and 4G8 (p<0.05 in post-hoc t-test after p<0.001 inANOVA). FIG. (H) illustrates that the strong staining of vasculardeposits of Aβ(arrows) occurs only with 6G1 and the commerciallyavailable antibody 6E10 while staining with 8F5 or 8C5 was much weaker.A qualitatively similar situation was found in Tg2576 mice (not shownhere).

FIG. 8 illustrates the selectivity of 8C5 for globulomers versusAβ(1-42) monomers, Aβ(1-40) and sAPP. Selectivity factors for 8C5 can becalculated as ratios between EC50 values (versus Aβ(1-42) monomer inHFIP: 2346/568.2=4.1; versus Aβ(1-42) monomer in NH₄OH: >100; versusAβ(1-40) monomer: >100; versus sAPP: >100)

FIG. 9(A) illustrates the nucleotide sequence (SEQ ID NO:11) encodingthe heavy chain of 8C5 and FIG. 9(B) illustrates the nucleotide sequence(SEQ ID NO:12) encoding the light chain of 8C5. The nucleotide sequencesencoding the corresponding CDRs, noted in FIGS. 10(A) and 10(B), areunderlined.

FIG. 10(B) illustrates the amino acid sequence (SEQ ID NO:19) of thevariable heavy chain of monoclonal antibody 8C5, and FIG. 10(B)illustrates the amino acid sequence (SEQ ID NO:20) of the variable lightchain of monoclonal antibody 8F5. One CDR of the variable heavy chain isrepresented by the amino acid sequence SYGMS (SEQ ID NO:13). Another CDRof the variable heavy chain is represented by the amino acid sequenceSIKNNGGSTYYPDSLKG (SEQ ID NO:14), and another CDR of the variable heavychain is represented by the amino acid sequence SGDY (SEQ ID NO:15). OneCDR of the variable light chain is represented by the amino acidsequence RSSQSLVHSNGDTFLH (SEQ ID NO:16). Another CDR of the variablelight chain is represented by the amino acid sequence KVSNRFS (SEQ IDNO:17), and another CDR of the variable light chain is represented bythe amino acid sequence SQSIHVPWT (SEQ ID NO:18). All of theabove-described CDRs are underlined in FIGS. 10(A) and 10(B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a monoclonal antibody, referred toherein as “8F5” as well as other related antibodies (e.g., 8C5), Theseantibodies may be used, for example, in the diagnosis, prevention andtreatment of Alzheimer's Diseases and other neurodegenerative disorders.

Monoclonal antibody 8F5 as well as monoclonal antibody 8C5 have manyinteresting properties which allow them to be extremely interestingtherapeutic candidates as well as extremely useful diagnosticcandidates. For example, monoclonal antibodies 8F5 and 8C5 havepreferential binding for Aβ(1-42) globulomers as compared with monomersor fibrils.

The term “Aβ(X-Y)” herein refers to the amino acid sequence from aminoacid position X to amino acid position Y of the human amyloid β proteinincluding both X and I and, in particular, refers to the amino acidsequence from amino acid position X to amino acid position Y of theamino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA orany of its naturally occurring variants, in particular, those with atleast one mutation selected from the group consisting of A2T, H6R, D7N,A21G (“Flemish”), E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”),D23N (“Iowa”), A42T and A42V wherein the numbers are relative to thestart position of the Aβ peptide, including both position X and positionY or a sequence with up to three additional amino acid substitutionsnone of which may prevent globulomer formation. An “additional” aminoacid substitution is defined herein as any deviation from the canonicalsequence that is not found in nature.

More specifically, the term “Aβ(1-42)” herein refers to the amino acidsequence from amino acid position 1 to amino acid position 42 of thehuman amyloid β protein including both 1 and 42 and, in particular,refers to the amino acid sequence from amino acid position 1 to aminoacid position 42 of the amino acid sequence DAEFRHDSGY EVHHQKLVFFAEDVGSNKGA IIGLMVGGVV IA (corresponding to amino acid positions 1 to 42)or any of its naturally occurring variants. Such variants may be, forexample, those with at least one mutation selected from the groupconsisting of A2T, HER, D7N, A21G (“Flemish”), E22G (“Arctic”), E22Q(“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V wherein thenumbers are relative to the start of the Aβ peptide, including both 1and 42 or a sequence with up to three additional amino acidsubstitutions none of which may prevent globulomer formation. Likewise,the term “Aβ(1-40)” here refers to the amino acid sequence from aminoacid position 1 to amino acid position 40 of the human amyloid β proteinincluding both 1 and 40 and refers, in particular, to the amino acidsequence from amino acid position 1 to amino acid position 40 of theamino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV or anyof its naturally occurring variants, Such variants include, for example,those with at least one mutation selected from the group consisting ofA2T, HER, D7N, A21G (“Flemish”), E22G (“Arctic”), E22Q (“Dutch”), E22K(“Italian”), and D23N (“Iowa”) wherein the numbers are relative to thestart position of the Aβ peptide, including both 1 and 40 or a sequencewith up to three additional amino acid substitutions none of which mayprevent globulomer formation.

The term “Aβ(X-Y) globulomer” (also known as “Aβ(X-Y) globularoligomer”) herein refers to a soluble, globular, non-covalentassociation of Aβ(X-Y) peptides, as defined above, possessinghomogeneity and distinct physical characteristics. The Aβ(X-Y)globulomers are stable, non-fibrillar, oligomeric assemblies of Aβ(X-Y)peptides which are obtainable by incubation with anionic detergents. Incontrast to monomer and fibrils, these globulomers are characterized bydefined assembly numbers of subunits (e.g., early assembly forms, n=3-6,oligomers A”, and late assembly forms, n=12-14, “oligomers B”, asdescribed in POT International Application Publication No. WO04/067561). The globulomers have a 3-dimensional globular type structure(“molten globule”, see Barghorn et al., 2005, J Neurochem, 95, 834-847).They may be further characterized by one or more of the followingfeatures:

-   cleavability of N-terminal amino acids X-23 with promiscuous    proteases (such as thermolysin or endoproteinase GluC) yielding    truncated forms Aβ(X-Y) globulomers;-   non-accessibility of C-terminal amino acids 24-Y with promiscuous    proteases and antibodies; and-   truncated forms of these Aβ(X-Y) globulomers maintain the    3-dimensional core structure of the globulomers with a better    accessibility of the core epitope Aβ(20-Y) in its globulomer    conformation.

According to the invention and, in particular, for the purpose ofassessing the binding affinities of the antibodies of the presentinvention, the term “Aβ(X-Y) globulomer” herein refers to a productwhich is obtainable by a process as described in InternationalApplication Publication No. WO 04/067561, which is incorporated hereinin its entirety by reference. The process comprises unfolding a natural,recombinant or synthetic Aβ(X-Y) peptide or a derivative thereof;exposing the at least partially unfolded Aβ(X-Y) peptide or derivativethereof to a detergent, reducing the detergent action and continuingincubation.

For the purpose of unfolding the peptide, hydrogen bond-breaking agentssuch as, for example, hexafluoroisopropanol (HFIP) may be allowed to acton the protein. Times of action of a few minutes, for example about 10to 60 minutes, are sufficient when the temperature of action is fromabout 20 to 50° C. and, in particular, about 35 to 40° C. Subsequentdissolution of the residue evaporated to dryness, preferably inconcentrated form, in suitable organic solvents miscible with aqueousbuffers such as, for example, dimethyl sulfoxide (DMSO), results in asuspension of the at least partially unfolded peptide or derivativethereof which can be used subsequently. If required, the stocksuspension may be stored at low temperature, for example, at about -20°C. for an interim period.

Alternatively, the peptide or the derivative thereof may be taken up inslightly acidic, preferably aqueous, solution, for example, a solutionof about 10 mM aqueous HCl. After an incubation time of approximately afew minutes, insoluble components are removed by centrifugation. A fewminutes at 10,000 g is expedient. These method steps are preferablycarried out at room temperature, i.e., a temperature in the range offrom 20 to 30° C. The supernatant obtained after centrifugation containsthe Aβ(X-Y) peptide or a derivative thereof and may be stored at lowtemperature, for example at about −20° C., for an interim period.

The following exposure to a detergent relates to oligomerization of thepeptide or the derivative thereof to give the intermediate type ofoligomers (in International Application Publication No. WO 04/067561referred to as oligomers A). For this purpose, a detergent is allowed toact on the, optionally, at least partially unfolded peptide orderivative thereof until sufficient intermediate oligomer has beenproduced. Preference is given to using ionic detergents, S inparticular, anionic detergents.

According to a particular embodiment, a detergent of the formula (I)

R-X,

is used, in which the radical “R” is unbranched or branched alkyl havingfrom 6 to 20 and preferably 10 to 14 carbon atoms or unbranched orbranched alkenyl having from 6 to 20 and preferably 10 to 14 carbonatoms, and the radical “X” is an acidic group or salt thereof with Xbeing preferably selected from among —COO⁻M⁺, —SO₃ ⁻M⁺ and is, mostpreferably, —OSO₃ ⁻M⁺; and M⁺ is a hydrogen cation or an inorganic ororganic cation preferably selected from alkali metal cations, alkalineearth metal cations and ammonium cations. Most advantageous aredetergents of the formula (I) in which R is an unbranched alkyl of whichalk-1-yl radicals must be mentioned, in particular. Particularpreference is given to sodium dodecyl sulfate (SDS). Lauric acid andoleic acid can also be used advantageously. The sodium salt of thedetergent lauroylsarcosin (also known as sarkosyl NL-30 or Gardol®) isalso particularly advantageous.

The time of detergent action, in particular, depends on whether, and ifyes, to what extent the peptide or derivative thereof subjected tooligomerization has unfolded. If, according to the unfolding step, thepeptide or derivative thereof has been treated beforehand with ahydrogen bond-breaking agent (i.e., in particular withhexafluoroisopropanol), times of action in the range of a few hours,advantageously from about 1 to 20 and, in particular, from about 2 to 10hours, are sufficient when the temperature of action is about 20 to 50°C. and, in particular, from about 35 to 40° C. If a less unfolded or anessentially not unfolded peptide or derivative thereof is the startingpoint, correspondingly longer times of action are expedient. If thepeptide or derivative thereof has been pretreated, for example,according to the procedure indicated above as an alternative to the HFIPtreatment or said peptide or derivative thereof is directly subjected tooligomerization, times of action in the range from about 5 to 30 hoursand, in particular, from about 10 to 20 hours are sufficient when thetemperature of action is from about 20 to 50° C. and, in particular,from about 35 to 40° C. After incubation, insoluble components areadvantageously removed by centrifugation, A few minutes at 10,000 g isexpedient.

The detergent concentration to be chosen depends on the detergent used.If SDS is used, a concentration in the range from 0.01 to 1% by weight,preferably, from 0.05 to 0.5% by weight, for example, of about 0.2% byweight, proves expedient. If lauric acid or oleic acid is used, somewhathigher concentrations are expedient, for example, in a range from 0.05to 2% by weight, preferably, from 0.1 to 0.5% by weight, for example, ofabout 0.5% by weight. The detergent action should take place at a saltconcentration approximately in the physiological range. Thus, inparticular NaCl concentrations in the range from 50 to 500 mM,preferably, from 100 to 200 mM and, more particularly, at about 140 mMare expedient.

The subsequent reduction of the detergent action and continuation ofincubation relates to further oligomerization to give the Aβ(X-Y)globulomer of the invention (in International Application PublicationNo. WO 04/067561 referred to as oligomer B). Since the compositionobtained from the preceding step regularly contains detergent and a saltconcentration in the physiological range, it is then expedient to reducedetergent action and, preferably, also salt concentration. This may becarried out by reducing the concentration of detergent and salt, forexample, by diluting expediently with water or a buffer of lower saltconcentration, for example, Tris-HCl, pH 7.3. Dilution factors in therange from about 2 to 10, advantageously, in the range from about 3 to 8and, in particular, of about 4, have proved suitable. The reduction indetergent action may also be achieved by adding substances which canneutralize this detergent action. Examples of these include substancescapable of complexing the detergents, like substances capable ofstabilizing cells in the course of purification and extraction measures,for example, particular EO/PO block copolymers, in particular, the blockcopolymer under the trade name Pluronic® F 68. Alkoxylated and, inparticular, ethoxylated alkyl phenols such as the ethoxylatedt-octylphenols of the Triton® X series, in particular, Triton® X100,3-(3-cholamidopropyldimethylammonio)-1-propanesulfonate (CHAPS®) oralkoxylated and, in particular, ethoxylated sorbitan fatty esters suchas those of the Tween® series, in particular, Tween® 20, inconcentration ranges around or above the particular critical micelleconcentration, may be equally used.

Subsequently, the solution is incubated until sufficient Aβ(X-Y)globulomer has been produced. Times of action in the range of severalhours, preferably, in the range from about 10 to 30 hours and, inparticular, in the range from about 15 to 25 hours, are sufficient whenthe temperature of action is about 20 to 50° C. and, in particular,about 35 to 40° C. The solution may then be concentrated and possibleresidues may be removed by centrifugation, Again, a few minutes at10,000 g proves expedient. The supernatant obtained after centrifugationcontains an Aβ(X-Y) globulomer as described herein.

An Aβ(X-Y) globulomer can be finally recovered, e.g. by ultrafiltration,dialysis, precipitation or centrifugation. It is further preferred ifelectrophoretic separation of the Aβ(X-Y) globulomers under denaturingconditions, e.g. by SDS-PAGE, produces a double band (e.g., with anapparent molecular weight of 38/48 kDa for Aβ(1-42)) and especiallypreferred if upon glutardialdehyde treatment of the oligomers, beforeseparation, these two bands are merged into one. It is also preferred ifsize exclusion chromatography of the globulomers results in a singlepeak (e.g., corresponding to a molecular weight of approximately 60 kDafor Aβ(1-42)). Starting from Aβ(1-42) peptide, the process is, inparticular, suitable for obtaining Aβ(1-42) globulomers

Preferably, the globulomer shows affinity to neuronal cells and alsoexhibits neuromodulating effects.

A “neuromodulating effect” is defined as a long-lasting inhibitoryeffect of a neuron leading to a dysfunction of the neuron with respectto neuronal plasticity.

According to another aspect of the invention, the term “Aβ(X-Y)globulomer” herein refers to a globulomer consisting essentially ofAβ(X-Y) subunits, wherein it is preferred if, on average, at least 11 of12 subunits are of the Aβ(X-Y) type, more preferred, if less than 10% ofthe globulomers comprise any non-Aβ(X-Y) peptides and, most preferred,if the content of non-Aβ(X-Y) peptides in the preparation is below thedetection threshold. More specifically, the term “Aβ(1-42) globulomer”herein refers to a globulomer comprising Aβ(1-42) units as definedabove; the term “Aβ(12-42) globulomer” herein refers to a globulomercomprising Aβ(12-42) units as defined above; and the term “Aβ(20-42)globulomer” herein refers to a globulomer comprising Aβ(20-42) units asdefined above.

The term “cross-linked Aβ(X-Y) globulomer” herein refers to a moleculeobtainable from an Aβ(X-Y) globulomer as described above bycross-linking, preferably, chemically cross-linking, more preferably,aldehyde cross-linking and, most preferably, glutardialdehydecross-linking of the constituent units of the globulomer. In anotheraspect of the invention, a cross-linked globulomer is essentially aglobulomer in which the units are at least partially joined by covalentbonds, rather than being held together by non-covalent interactionsonly.

The term “Aβ(X-Y) globulomer derivative” herein refers, in particular,to a globulomer that is labelled by being covalently linked to a groupthat facilitates detection, preferably, a fluorophore, e.g., fluoresceinisothiocyanate, phycoerythrin, Aequorea victoria fluorescent protein,Dictyosoma fluorescent protein or any combination or fluorescence-activederivatives thereof; a chromophore; a chemoluminophore, e.g.,luciferase, preferably Photinus pyralis luciferase, Vibrio fischeriluciferase, or any combination or chemoluminescence-active derivativesthereof; an enzymatically active group, e.g., peroxidase such ashorseradish peroxidase, or an enzymatically active derivative thereof;an electron-dense group, e.g., a heavy metal containing group such as agold containing group; a hapten, e.g., a phenol derived hapten; astrongly antigenic structure, e.g., peptide sequence predicted to beantigenic such as by the algorithm of Kolaskar and Tongaonkar; anaptamer for another molecule; a chelating group, e.g., hexahistidinyl; anatural or nature-derived protein structure mediating further specificprotein-protein interactions, e.g., a member of the fos/jun pair; amagnetic group, e.g., a ferromagnetic group; or a radioactive group suchas a group comprising ¹H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I or any combinationthereof; or to a globulomer flagged by being covalently or bynon-covalently linked by high-affinity interaction, preferably,covalently linked to a group that facilitates inactivation,sequestration, degradation and/or precipitation, preferably, flaggedwith a group that promotes in vivo degradation, more preferably, withubiquitin, where it is particularly preferred if this flagged oligomeris assembled in vivo; or to a globulomer modified by any combination ofthe above. Such labelling and flagging groups and methods for attachingthem to proteins are known in the art. Labelling and/or flagging may beperformed before, during or after globulomerization. In another aspectof the invention, a globulomer derivative is a molecule obtainable froma globulomer by a labelling and/or flagging reaction. Correspondingly,the term “Aβ(X-Y) monomer derivative” herein refers, in particular, toan Aβ monomer that is labelled or flagged as described for theglobulomer.

The term “greater affinity” herein refers to a degree of interactionwhere the equilibrium between unbound antibody and unbound globulomer,on the one hand, and antibody-globulomer complex, on the other, isfurther in favor of the antibody-globulomer complex. Likewise, the term“smaller affinity” herein refers to a degree of interaction where theequilibrium between unbound antibody and unbound globulomer, on the onehand, and antibody-globulomer complex, on the other, is further in favorof the unbound antibody and unbound globulomer.

The term “Aβ(X-Y) monomer” herein refers to the isolated form of theAβ(X-Y) peptide, preferably, a form of the Aβ(X-Y) peptide which is notengaged in essentially non-covalent interactions with other Aβ peptides.Practically, the Aβ(X-Y) monomer is usually provided in the form of anaqueous solution. Preferably, the aqueous monomer solution contains0.05% to 0.2%, more preferably, about 0.1% NaOH when used, for instance,for determining the binding affinity of the antibody of the presentinvention. In another preferable situation, the aqueous monomer solutioncontains 0.05% to 0.2%, more preferably, about 0.1% NaOH. When used, itmay be expedient to dilute the solution in an appropriate manner.Further, it is usually expedient to use the solution within 2 hours, inparticular, within 1 hour, and, especially, within 30 minutes after itspreparation.

The term “fibril” herein refers to a molecular structure that comprisesassemblies of non-covalently associated, individual Aβ(X-Y) peptideswhich show fibrillary structure under the electron microscope, whichbind Congo red, exhibit birefringence under polarized light and whoseX-ray diffraction pattern is a cross-β structure. The fibril may also bedefined as a molecular structure obtainable by a process that comprisesthe self-induced polymeric aggregation of a suitable Aβ peptide in theabsence of detergents, e.g., in 0.1 M HCl, leading to the formation ofaggregates of more than 24, preferably, more than 100 units. Thisprocess is well known in the art. Expediently, Aβ(X-Y) fibril is used inthe form of an aqueous solution. In a particularly preferred embodimentof the invention, the aqueous fibril solution is made by dissolving theAβ peptide in 0.1% NH₄OH, diluting it 1:4 with 20 mM NaH₂PO₄, 140 mMNaCl, pH 7.4, followed by readjusting the pH to 7.4, incubating thesolution at 37° C. for 20 h, followed by centrifugation at 10000 g for10 min and resuspension in 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4.

The term “Aβ(X-Y) fibril” herein refers to a fibril comprising Aβ(X-Y)subunits where it is preferred if, on average, at least 90% of thesubunits are of the Aβ(X-Y) type, more preferred, if at least 98% of thesubunits are of the Aβ(X-Y) type and, most preferred, if the content ofnon-Aβ(X-Y) peptides is below the detection threshold.

Turning back to 8F5, as evidenced by FIG. 1, as well as 8C5 (FIG. 8),Aβ(1-42) globulomer-specific antibodies monoclonal antibodies 8F5 and8C5 recognize predominantly Aβ(1-42) globulomer forms and not standardpreparations of Aβ(1-40) or Aβ(1-42) monomers including aggregatedAβ(1-42) in contrast to nonspecific antibodies 6G1 and 6E10. Inparticular, 8F5 detects Aβ(1-42) globulomers only by native PAGE-westernblot and not by SDS-PAGE Western blot analysis indicating binding to amore complex detergent-dissociable intersubunit epitope in the coreAβ(1-42) globulomer structure. An intersubunit epitope is defined as acomplex non-linear through space epitope located on at least twosubunits. More specifically, dot-blot analysis against various Aβ(1-42)and Aβ(1-40) standard preparations showed significant differences inrecognition of Aβ(1-42) globulomer versus non-globulomer Ap forms(standard Aβ(1-40)/(1-42) monomer preparation, aggregated Aβ(1-42) forspecific 8F5 and 8C5 but not for the isoform non-specific antibodies 6G1and 6E10. The globulomer specificity of 8F5 and 8C5 but not of 6G1 and6E10, was confirmed by quantifying Aβ(1-42) globulomer, Aβ(1-42)monomer, Aβ(1-40) monomer and soluble amyloid precursor protein alphabinding in sandwich ELISAs. Further, since these antibodies access theglobulomer after native but not after SDS Western blotting, it is likelythat each antibody recognizes a structural non-linear epitope in betweensubunits in the region of amino acids 20 to 30 of Aβ(1-42). Suchspecificity for globulomers is important because specifically targetingthe globulomer form of Aβ with a globulomer preferential antibody suchas, for example, 8F5 or 8C5 will: 1) avoid targeting insoluble amyloiddeposits, binding to which may account for inflammatory side effectsobserved during immunizations with insoluble Aβ; 2) spare Aβ monomer andAPP that are reported to have precognitive physiological functions (Planet al., J. of Neuroscience 23:5531-5535 (2003); and 3) increase thebioavailability of the antibody, as it would not be shaded orinaccessible through extensive binding to insoluble deposits.

The subject invention also includes isolated nucleotide sequences (orfragments thereof) encoding the variable light and heavy chains ofmonoclonal antibody 8F5 and 8CD as well as those nucleotide sequences(or fragments thereof) having sequences comprising, corresponding to,identical to, hybridizable to, or complementary to at least about 70%(e.g,, 70% 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%), preferably atleast about 80% (e,g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or89%), and more preferably at least about 90% (e.g, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%) identity to these encoding nucleotidesequences. (All integers (and portions thereof) between and including70% and 100% are considered to be within the scope of the presentinvention with respect to percent identity.) Such sequences may bederived from any source (e.g., either isolated from a natural source,produced via a semi-synthetic route, or synthesized de novo). Inparticular, such sequences may be isolated or derived from sources otherthan described in the examples (e.g., bacteria, fungus, algae, mouse orhuman),

In addition to the nucleotide sequences described above, the presentinvention also includes amino acid sequences of the variable light andheavy chains of monoclonal antibody 8F5 and monoclonal antibody 8C5 (orfragments of these amino acid sequences). Further, the present inventionalso includes amino acid sequences (or fragments thereof) comprising,corresponding to, identical to, or complementary to at least about 70%,preferably at least about 80%, and more preferably at least about 90%identity to the amino acid sequences of the proteins of the presentinvention. (Again, all integers (and portions thereof) between andincluding 70% and 100% (as recited in connection with the nucleotidesequence identities noted above) are also considered to be within thescope of the present invention with respect to percent identity.)

For purposes of the present invention, a “fragment” of a nucleotidesequence is defined as a contiguous sequence of approximately at least6, preferably at least about 8, more preferably at least about 10nucleotides, and even more preferably at least about 15 nucleotidescorresponding to a region of the specified nucleotide sequence.

The term “identity” refers to the relatedness of two sequences on anucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments (or two amino acid sequences).“Percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over a particular region, determining thenumber of positions at which the identical base or amino acid occurs inboth sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the segment being compared and multiplying the result by 100. Optimalalignment of sequences may be conducted by the algorithm of Smith &Waterman, Appl. Math. 2:482 (1981), by the algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the method of Pearson & Lipman,Proc. Natl. Acad. Sci. (USA) 85:2444 (1988) and by computer programswhich implement the relevant algorithms (e.g., Clustal Macaw Pileup(http://cmgm.stanford.edu/biochem218/11Multiple.pdf; Higgins et al.,CABIOS. 5L151-153 (1989)), FASTDB (Intelligenetics), BLAST (NationalCenter for Biomedical Information; Altschul et al., Nucleic AcidsResearch 25:3389-3402 (1997)), PILEUP (Genetics Computer Group, Madison,Wis.) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, Madison, Wis.). (See U.S.Pat. No. 5,912,120.)

For purposes of the present invention, “complementarity” is defined asthe degree of relatedness between two DNA segments. It is determined bymeasuring the ability of the sense strand of one DNA segment tohybridize with the anti-sense strand of the other DNA segment, underappropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand.

In the double helix, adenine appears in one strand, thymine appears inthe other strand. Similarly, wherever guanine is found in one strand,cytosine is found in the other. The greater the relatedness between thenucleotide sequences of two DNA segments, the greater the ability toform hybrid duplexes between the strands of the two DNA segments.

“Similarity” between two amino acid sequences is defined as the presenceof a series of identical as well as conserved amino acid residues inboth sequences. The higher the degree of similarity between two aminoacid sequences, the higher the correspondence, sameness or equivalenceof the two sequences. (“Identity between two amino acid sequences isdefined as the presence of a series of exactly alike or invariant aminoacid residues in both sequences.) The definitions of “complementarity”,“identity” and “similarity” are well known to those of ordinary skill inthe art.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 amino acids, morepreferably at least 8 amino acids, and even more preferably at least 15amino acids from a polypeptide encoded by the nucleic acid sequence.

Additionally, a nucleic acid molecule is “hybridizable” to anothernucleic acid molecule when a single-stranded form of the nucleic acidmolecule can anneal to the other nucleic acid molecule under theappropriate conditions of temperature and ionic strength (see Sambrooket al., “Molecular Cloning: A Laboratory Manual, Second Edition (1989),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization.

The term “hybridization” as used herein is generally used to meanhybridization of nucleic acids at appropriate conditions of stringencyas would be readily evident to those skilled in the art depending uponthe nature of the probe sequence and target sequences. Conditions ofhybridization and washing are well known in the art, and the adjustmentof conditions depending upon the desired stringency by varyingincubation time, temperature and/or ionic strength of the solution arereadily accomplished. See, for example, Sambrook, J, et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold spring harbor Press,Cold Spring harbor, N.Y., 1989, as noted above and incorporated hereinby reference. (See also Short Protocols in Molecular Biology, ed.Ausubel et al. and Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays” (1993), bothincorporated herein by reference.) Specifically, the choice ofconditions is dictated by the length of the sequences being hybridized,in particular, the length of the probe sequence, the relative G-Ccontent of the nucleic acids and the amount of mismatches to bepermitted. Low stringency conditions are preferred when partialhybridization between strands that have lesser degrees ofcomplementarity is desired. When perfect or near perfect complementarityis desired, high stringency conditions are preferred, For typical highstringency conditions, the hybridization solution contains 6×S.S.C.,0.01 M EDTA, 1× Denhardt's solution and 0.5% SDS. Hybridization iscarried out at about 68 degrees Celsius for about 3 to 4 hours forfragments of cloned DNA and for about 12 to about 16 hours for totaleukaryotic DNA. For moderate stringencies, one may utilize filterpre-hybridizing and hybridizing with a solution of 3×sodium chloride,sodium citrate (SSC), 50% formamide (0.1 M of this buffer at pH 7.5) and5×Denhardt's solution. One may then pre-hybridize at 37 degrees Celsiusfor 4 hours, followed by hybridization at 37 degrees Celsius with anamount of labeled probe equal to 3,000,000 cpm total for 16 hours,followed by a wash in 2×SSC and 0.1% SDS solution, a wash of 4 times for1 minute each at room temperature and 4 times at 60 degrees Celsius for30 minutes each. Subsequent to drying, one exposes to film. For lowerstringencies, the temperature of hybridization is reduced to about 12degrees Celsius below the melting temperature (T_(m)) of the duplex. TheT_(m) is known to be a function of the G-C content and duplex length aswell as the ionic strength of the solution.

“Hybridization” requires that two nucleic acids contain complementarysequences. However, depending on the stringency of the hybridization,mismatches between bases may occur.. As noted above, the appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation. Such variables are wellknown in the art. More specifically, the greater the degree ofsimilarity or homology between two nucleotide sequences, the greater thevalue of Tm for hybrids of nucleic acids having those sequences, Forhybrids of greater than 100 nucleotides in length, equations forcalculating Tm have been derived (see Sambrook et al., supra). Forhybridization with shorter nucleic acids, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra).

As used herein, an “isolated nucleic acid fragment or sequence” is apolymer of RNA or DNA that is single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases. Anisolated nucleic acid fragment in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.(A “fragment” of a specified polynucleotide refers to a polynucleotidesequence which comprises a contiguous sequence of approximately at leastabout 6 nucleotides, preferably at least about 8 nucleotides, morepreferably at least about 10 nucleotides, and even more preferably atleast about 15 nucleotides, and most preferable at least about 25nucleotides identical or complementary to a region of the specifiednucleotide sequence.) Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

The terms “fragment or subfragment that is functionally equivalent” and“functionally equivalent fragment or subfragment” are usedinterchangeably herein. These terms refer to a portion or subsequence ofan isolated nucleic acid fragment in which the ability to alter geneexpression or produce a certain phenotype is retained whether or not thefragment or subfragment encodes an active enzyme. For example, thefragment or subfragment can be used in the design of chimeric constructsto produce the desired phenotype in a transformed plant. Chimericconstructs can be designed for use in co-suppression or antisense bylinking a nucleic acid fragment or subfragment thereof, whether or notit encodes an active enzyme, in the appropriate orientation relative toa plant promoter sequence.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein, Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.

“Native gene” refers to a gene as found in nature with its ownregulatory sequences. In contrast,“chimeric construct” refers to acombination of nucleic acid fragments that are not normally foundtogether in nature. Accordingly, a chimeric construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than thatnormally found in nature. (The term “isolated” means that the sequenceis removed from its natural environment.)

A “foreign” gene refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric constructs. A “transgene” is a genethat has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

“Promoter” or “regulatory gene sequence” refers to a DNA sequencecapable of controlling the expression of a coding sequence or functionalRNA. The sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence which can stimulatepromoter or regulatory gene sequence activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoter sequences canalso be located within the transcribed portions of genes, and/ordownstream of the transcribed sequences. Promoters may be derived intheir entirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most host cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, Biochemistry of Plants 15:1-82 (1989). It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

An “intron” is an intervening sequence in a gene that does not encode aportion of the protein sequence. Thus, such sequences are transcribedinto RNA but are then excised and are not translated. The term is alsoused for the excised RNA sequences. An “exon” is a portion of the genesequence that is transcribed and is found in the mature messenger RNAderived from the gene, but is not necessarily a part of the sequencethat encodes the final gene product.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., Plant Cell1:671-680 (1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the Klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript that includes the mRNA and can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to an RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence,“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but yet has an effect on cellular processes.The terms “complement” and “reverse complement” are used interchangeablyherein with respect to mRNA transcripts, and are meant to define theantisense RNA of the message.

The term “endogenous RNA” refers to any RNA which is encoded by anynucleic acid sequence present in the genome of the host prior totransformation with the recombinant construct of the present invention,whether naturally-occurring or non-naturally occurring, i.e., introducedby recombinant means, mutagenesis, etc.

The term “non-naturally occurring” means artificial, not consistent withwhat is normally found in nature.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression of a gene involves transcription ofthe gene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and pro-peptidesstill present. Pre- and pro-peptides may be but are not limited tointracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, resulting in geneticallystable inheritance. In contrast, “transient transformation” refers tothe transfer of a nucleic acid fragment into the nucleus, orDNA-containing organelle, of a host organism resulting in geneexpression without integration or stable inheritance. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” organisms. The term “transformation” as used herein refersto both stable transformation and transient transformation.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T,, Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

Polymerase chain reaction (“PCR”) is a powerful technique used toamplify DNA millions of fold, by repeated replication of a template, ina short period of time. (Mullis et al., Cold Spring Harbor Symp. Quant.Biol. 51:263-273 (1986); Erlich et al., European Patent Application No.50,424; European Patent Application No. 84,796; European PatentApplication No. 258,017; European Patent Application No. 237,362;Mullis, European Patent Application No. 201,184; Mullis et al., U.S.Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki et al.,U.S. Pat. No. 4,683,194). The process utilizes sets of specific in vitrosynthesized oligonucleotides to prime DNA synthesis. The design of theprimers is dependent upon the sequences of DNA that are to be analyzed.The technique is carried out through many cycles (usually 20-50) ofmelting the template at high temperature, allowing the primers to annealto complementary sequences within the template and then replicating thetemplate with DNA polymerase.

The products of PCR reactions are analyzed by separation in agarose gelsfollowed by ethidium bromide staining and visualization with UVtransillumination, Alternatively, radioactive dNTPs can be added to thePCR in order to incorporate label into the products. In this case theproducts of PCR are visualized by exposure of the gel to x-ray film. Theadded advantage of radiolabeling PCR products is that the levels ofindividual amplification products can be quantitated.

The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be itself or may be usedin conjunction with a vector. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art. For example, aplasmid can be used. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells comprising any of theisolated nucleic acid fragments of the invention. The skilled artisanwill also recognize that different independent transformation eventswill result in different levels and patterns of expression (Jones etal., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

A “monoclonal antibody” as used herein is intended to refer to one of apreparation of antibody molecules containing antibodies which share acommon heavy chain and common light chain amino acid sequence, incontrast with an antibody from a “polyclonal” antibody preparation whichcontains a mixture of different antibodies. Monoclonal antibodies can begenerated by several novel technologies like phage, bacteria, yeast orribosomal display, as well as classical methods exemplified byhybridoma-derived antibodies (e.g., an antibody secreted by a hybridomaprepared by hybridoma technology, such as the standard Kohler andMilstein hybridoma methodology ((1975) Nature 256:495-497). Thus, anon-hybridoma-derived agonistic antibody of the invention is stillreferred to as a monoclonal antibody although it may have been derivedby non-classical methodologies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to a globulomer is substantially free of antibodies thatspecifically bind antigens other than a globulomer). An isolatedantibody that specifically binds a globulomer may, however, havecross-reactivity to other antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which comprises a singlevariable domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al, (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides, Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies, Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library(Hoogenboom H, R., (1997) TIB Tech. 15:62-70; Azzazy H., and HighsmithW. E., (2002) Clin, Biochem. 35:425-445; Gavilondo J. V., and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000)Immunology Today 21:371-378), antibodies isolated from an animal (e.g.,a mouse) that is transgenic for human immunoglobulin genes (see e.g.,Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; KellermannS-A., and Green L. L. (2002) Current Opinion in Biotechnology13:593-597; Little M. et al (2000) Immunology Today 21:364-370) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo. (See also Kabat at al. Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, 1991), The humanantibodies of the present invention, however, may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in viva). (See also Harlow and Lane, Antibodies: ALaboratory Manual, New York: Cold Spring Harbor Press, 1990).

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of V_(H) and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

Recombinant human antibodies of the present invention have variableregions, and may also include constant regions, derived from humangermline immunoglobulin sequences. (See Kabat et al, (1991) supra.) Incertain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis), and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo. In certain embodiments, however,such recombinant antibodies are the result of selective mutagenesis orbackmutation or both.

The term “backmutation” refers to a process in which some or all of thesomatically mutated amino acids of a human antibody are replaced withthe corresponding germline residues from a homologous germline antibodysequence. The heavy and light chain sequences of a human antibody of theinvention are aligned separately with the germline sequences in theVBASE database to identify the sequences with the highest homology.VBASE is a comprehensive directory of all human germline variable regionsequences compiled from published sequences, including current releasesof GenBank and EMBL data libraries. The database has been developed atthe MRC Centre for Protein Engineering (Cambridge, UK) as a depositoryof the sequenced human antibody genes (website:http://www.mrc-cpe.cam.ac.uk/vbase-intro.php?menu=901). Differences inthe human antibody of the invention are returned to the germlinesequence by mutating defined nucleotide positions encoding suchdifferent amino acids. The role of each amino acid thus identified as acandidate for backmutation should be investigated for a direct orindirect role in antigen binding, and any amino acid found aftermutation to affect any desirable characteristic of the human antibodyshould not be included in the final human antibody. To minimize thenumber of amino acids subject to backmutation, those amino acidpositions found to be different from the closest germline sequence, butidentical to the corresponding amino acid in a second germline sequence,can remain, provided that the second germline sequence is identical andco-linear to the sequence of the human antibody of the invention for atleast 10, preferably 12, amino acids on both sides of the amino acid inquestion. Backmutation may occur at any stage of antibody optimization.

A “labeled binding protein” is a protein wherein an antibody or antibodyportion of the invention is derivatized or linked to another functionalmolecule (e.g., another peptide or protein). For example, a labeledbinding protein of the invention can be derived by functionally linkingan antibody or antibody portion of the invention (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

For purposes of the present invention, a “glycosylated binding protein”comprises a protein wherein the antibody or antigen-binding portionthereof comprises one or more carbohydrate residues. Nascent in vivoprotein production may undergo further processing, known aspost-translational modification. In particular, sugar (glycosyl)residues may be added enzymatically, a process known as glycosylation.The resulting proteins bearing covalently linked oligosaccharide sidechains are known as glycosylated proteins or glycoproteins. Antibodiesare glycoproteins with one or more carbohydrate residues in the Fcdomain, as well as the variable domain. Carbohydrate residues in the Fcdomain have important effect on the effector function of the Fc domain,with minimal effect on antigen binding or half-life of the antibody (R.Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast,glycosylation of the variable domain may have an effect on the antigenbinding activity of the antibody. Glycosylation in the variable domainmay have a negative effect on antibody binding affinity, likely due tosteric hindrance (Co, M. S., et al., Mol, Immunol. (1993) 30:1361-1367),or result in increased affinity for the antigen (Wallick, S. C., et al.,Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991)10:2717 2723). Further, glycosylation site mutants can be made in whichthe O- or N-linked glycosylation site of the binding protein has beenmutated. One skilled in the art can generate such mutants using standardwell-known technologies. Glycosylation site mutants that retain thebiological activity but have increased or decreased binding activity arealso contemplated.

Further, the glycosylation of the antibody or antigen-binding portion ofthe invention can modified. For example, an aglycoslated antibody can bemade (i.e., the antibody lacks glycosylation). Glycosylation can bealtered to, for example, increase the affinity of the antibody forantigen. Such carbohydrate modifications can be accomplished by, forexample, altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region glycosylationsites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen.Such an approach is described in further detail in InternationalApplication Publication No. WO 03/016466A2, and U.S. Pat. Nos. 5,714,350and 6,350,861, each of which is incorporated herein by reference in itsentirety.

Additionally or alternatively, a modified antibody can be made that hasan altered type of glycosylation, such as a hypofucosylated antibodyhaving reduced amounts of fucosyl residues or an antibody havingincreased bisecting GlcNAc structures. Such altered glycosylationpatterns have been demonstrated to increase the ADCC ability ofantibodies. Such carbohydrate modifications can be accomplished by, forexample, expressing the antibody in a host cell with alteredglycosylation machinery. Cells with altered glycosylation machinery havebeen described in the art and can be used as host cells in which toexpress recombinant antibodies of the invention to thereby produce anantibody with altered glycosylation. (See, for example, Shields, R. L.et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al., (1999) Nat.Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195;International Application Publication Number WO 03/035835 and WO99/5434280, each of which is incorporated herein by reference in itsentirety.)

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity, Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S Patent ApplicationPublication Nos. 20040018590 and 20020137134 and InternationalApplication Publication No, WO 05/100584 A2).

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

The invention also provides a method for making the monoclonalantibodies of the invention from non-human, non-mouse animals byimmunizing non-human transgenic animals that comprise humanimmunoglobulin loci. One may produce such animals using methods known inthe art. In a preferred embodiment, the non-human animals may be rats,sheep, pigs, goats, cattle or horses. Antibody-producing immortalizedhybridomas may be prepared from the immunized animal. Afterimmunization, the animal is sacrificed and the splenic B cells are fusedto immortalized myeloma cells as is well known in the art. See, e.g.,Harlow and Lane, supra. In a preferred embodiment, the myeloma cells donot secrete immunoglobulin polypeptides (a non-secretory cell line).After fusion and antibiotic selection, the hybridomas are screened usingan antigen (for example, a globulomer) or a portion thereof, or a cellexpressing the antigen of interest. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in International Application Publication No.WO 00/37504, herein incorporated by reference.

The antibody-producing hybridomas are selected, cloned and furtherscreened for desirable characteristics, including robust hybridomagrowth, high antibody production and desirable antibody characteristics,as discussed further below. Hybridomas may be cultured and expanded invivo in syngeneic animals, in animals that lack an immune system, e.g.,nude mice, or in cell culture in vitro. Methods of selecting, cloningand expanding hybridomas are well known to those of ordinary skill inthe art. Preferably, the immunized animal is a non-human animal thatexpresses human immunoglobulin genes and the splenic B cells are fusedto a myeloma derived from the same species as the non-human animal.

In one aspect, the invention provides hybridomas that produce monoclonalantibodies to be used in the treatment, diagnosis and prevention ofAlzheimer's Disease. In a preferred embodiment, the hybridomas are mousehybridomas. In another preferred embodiment, the hybridomas are producedin a non-human, non-mouse species such as rats, sheep, pigs, goats,cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an antibody against a globulomer.

Recombinant antibodies may be generated from single, isolatedlymphocytes using a procedure referred to in the art as the selectedlymphocyte antibody method (SLAM), as described in U.S. Pat. No.5,627,052, International Application Publication No. WO 92/02551 andBabcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. Inthis method, single cells secreting antibodies of interest (e.g.,lymphocytes derived from the immunized animal) are screened using anantigen-specific hemolytic plaque assay, wherein the antigen (e.g.,globulomer), or a fragment thereof, is coupled to sheep red blood cellsusing a linker, such as biotin, and used to identify single cells thatsecrete antibodies with specificity for the antigen. Followingidentification of antibody-secreting cells of interest, heavy- andlight-chain variable region cDNAs are rescued from the cells by reversetranscriptase-POR and these variable regions can then be expressed, inthe context of appropriate immunoglobulin constant regions (e.g., humanconstant regions), in mammalian host cells, such as COS or CHO cells.The host cells transfected with the amplified immunoglobulin sequences,derived from in vivo selected lymphocytes, can then undergo furtheranalysis and selection in vitro, for example by panning the transfectedcells to isolate cells expressing antibodies to IL-18. The amplifiedimmunoglobulin sequences further can be manipulated in vitro, such as byin vitro affinity maturation methods such as those described inInternational Application Publication No. WO 97/29131 and InternationalApplication Publication No. WO 00/56772.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a nonhuman species (e.g.,a mouse) but in which at least a portion of the VH and/or VL sequencehas been altered to be more “human-like”, i.e., more similar to humangermline variable sequences, One type of humanized antibody is aCDR-grafted antibody in which human CDR sequences are introduced intononhuman VH and VL sequences to replace the corresponding nonhuman CDRsequences. In particular, the term “humanized antibody” is an antibodyor a variant, derivative, analog or fragment thereof whichimmunospecifically binds to an antigen of interest and which comprises aframework (FR) region having substantially the amino acid sequence of ahuman antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 80%, preferably at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identical to theamino acid sequence of a non-human antibody CDR. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence.Preferably, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin, In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain, The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In other embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. Further, as used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen,

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BlAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “K_(on)”, as used herein, is intended to refer to the “on rate”constant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “K_(off)”, as used herein, is intended to refer to the “offrate” constant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(d)”, as used herein, is intended to refer to the“dissociation constant” of a particular antibody-antigen interaction asis known in the art.

The term “labeled binding protein” as used herein, refers to a proteinwith a label incorporated that provides for the identification of thebinding protein. Preferably, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho or ¹⁵³Sm); fluorescent labels(e.g., FITC, rhodamine or lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase or alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal binding domainsor epitope tags); and magnetic agents, such as gadolinium chelates.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably, the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin, as well as analogs and homologs of theseagents.

The terms “crystal”, and “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter.

The term “immunize” refers herein to the process of presenting anantigen to an immune repertoire whether that repertoire exists in anatural genetically unaltered organism, or a transgenic organismmodified to display an artificial human immune repertoire. Similarly, an“immunogenic preparation” is a formulation of antigen that containsadjuvants or other additives that would enhance the immunogenicity ofthe antigen. An example of this would be co-injection of a purified formof GLP-1 receptor with Freund's complete adjuvant into a mouse.“Hyperimmunization”, as defined herein, is the act of serial, multiplepresentations of an antigen in an immunogenic preparation to a hostanimal with the intention of developing a strong immune response.

One way of measuring the binding kinetics of an antibody is by surfaceplasmon resonance. The term “surface plasmon resonance”, as used herein,refers to an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBiacore system (Biacore International, Upsala, Sweden and Piscataway,N.J.). For further descriptions, see Jönsson et al. (1993) Annales deBiologie Clinique (Paris) 51:19-26; Jönsson et al. (1991) Biotechniques11:620-627; Johnsson et al. (1995) Journal of Molecular Recognition8:125-131; and Johnnson et al. (1991) Analytical Biochemistry198:268-277.

A “pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention, A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects, A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for, forexample, parenteral administration. Preferably, the antibody orantibody-portions will be prepared as an injectable solution containing0.1-250 mg/ml antibody. The injectable solution can be composed ofeither a liquid or lyophilized dosage form in a flint or amber vial,ampule or pre-filled syringe. The buffer can be L-histidine (1-50 mM),optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitablebuffers include but are not limited to, sodium succinate, sodiumcitrate, sodium phosphate or potassium phosphate. Sodium chloride can beused to modify the toxicity of the solution at a concentration of 0-300mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can beincluded for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trenhaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-Methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-BO (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRIJ surfactants,

The compositions of this invention may be in a variety of forms, Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid, Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art, See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingAlzheimer's Disease or related diseases or conditions. For example, oneof the antibodies of the subject invention or antibody portion thereofmay be coformulated and/or coadministered with one or more additionalantibodies that bind other targets.

In certain embodiments, a monoclonal antibody of the subject inventionor fragment thereof may be linked to a half-life extending vehicle knownin the art. Such vehicles include, but are not limited to, the Fcdomain, polyethylene glycol, and dextran. Such vehicles are described,e.g., in U.S. application Ser. No. 09/428,082 and published PCTApplication No, WO 99/25044, which are hereby incorporated by referencefor any purpose.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al,, Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Plant Molecular Biology: A LaboratoryManual, eds, Clark, Springer, N.Y. (1997)).

Uses of the Monoclonal Antibody

The monoclonal antibodies of the present invention (e.g., 8F5 and 8CF)have many interesting utilities. For example, the monoclonal antibodiesmay be used in the prevention, treatment and diagnosis of Alzheimer'sDisease as described above. Further, the antibodies may be used in thedevelopment of anti-antibodies. Further, the hybridoma producing therespective antibody allows for the steady production of a continuoussource of identical monoclonal antibodies (i.e., reagents), therebyguaranteeing identity between antibodies in various experiments as wellas therapeutic uses.

Also, the methods of the present invention allow one to prepareappropriate amounts of starting material for use in the preparation offurther materials that, in turn, may be utilized in the production ofmonoclonal antibodies (or other antibodies) for the treatment ofAlzheimer's Disease. As noted above, the antibodies may also be used forpassive immunization in order to prevent Alzheimer's Disease or otherrelated neurological conditions characterized by the same symptoms asAlzheimer's Disease such as cognitive impairment.

In one diagnostic embodiment of the present invention, an antibody ofthe present invention (e.g., 8F5), or a portion thereof, is coated on asolid phase (or is present in a liquid phase). The test or biologicalsample (e.g., whole blood, cerebrospinal fluid, serum, etc.) is thencontacted with the solid phase. If antigen (e.g., globulomer) is presentin the sample, such antigens bind to the antibodies on the solid phaseand are then detected by either a direct or indirect method. The directmethod comprises simply detecting presence of the complex itself andthus presence of the antigens. In the indirect method, a conjugate isadded to the bound antigen. The conjugate comprises a second antibody,which binds to the bound antigen, attached to a signal-generatingcompound or label. Should the second antibody bind to the bound antigen,the signal-generating compound generates a measurable signal. Suchsignal then indicates presence of the antigen in the test sample.

Examples of solid phases used in diagnostic immunoassays are porous andnon-porous materials, latex particles, magnetic particles,microparticles (see e.g., U.S. Pat. No. 5,705,330), beads, membranes,microtiter wells and plastic tubes. The choice of solid phase materialand method of labeling the antigen or antibody present in the conjugate,if desired, are determined based upon desired assay format performancecharacteristics.

As noted above, the conjugate (or indicator reagent) will comprise anantibody (or perhaps anti-antibody, depending upon the assay), attachedto a signal-generating compound or label. This signal-generatingcompound or “label” is itself detectable or may be reacted with one ormore additional compounds to generate a detectable product. Examples ofsignal-generating compounds include chromogens, radioisotopes (e.g.,125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g.,acridinium), particles (visible or fluorescent), nucleic acids,complexing agents, or catalysts such as enzymes (e.g., alkalinephosphatase, acid phosphatase, horseradish peroxidase,beta-galactosidase and ribonuclease). In the case of enzyme use (e.g.,alkaline phosphatase or horseradish peroxidase), addition of a chromo-,fluro-, or lump-genic substrate results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, amplification (e.g., polymerase chainreaction) and Raman spectroscopy are also useful.

Examples of biological fluids which may be tested by the aboveimmunoassays include plasma, whole blood, dried whole blood, serum,cerebrospinal fluid or aqueous or organo-aqueous extracts of tissues andcells.

The present invention also encompasses a method for detecting thepresence of antibodies in a test sample. This method comprises the stepsof: (a) contacting the test sample suspected of containing antibodieswith anti-antibody specific for the antibodies in the patient sampleunder time and conditions sufficient to allow the formation ofanti-antibody/antibody complexes, wherein the anti-antibody is anantibody of the present invention which binds to an antibody in thepatient sample; (b) adding a conjugate to the resultinganti-antibody/antibody complexes, the conjugate comprising an antigen(which binds to the anti-antibody) attached to a signal generatingcompound capable of detecting a detectable signal; and (d) detecting thepresence of the antibodies which may be present in the test sample bydetecting the signal generated by the signal generating compound, Acontrol or calibrator may be used which comprises antibody to theanti-antibody.

The present invention also includes a vaccine comprising one of more ofthe antibodies described herein or a portion thereof and apharmaceutically acceptable adjuvant (e.g., Freund's adjuvant orphosphate buffered saline).

Kits are also included within the scope of the present invention. Morespecifically, the present invention includes kits for determining thepresence of antigens (e,g., globulomers) in a patient suspected ofhaving Alzheimer's

Disease or another condition characterized by cognitive impairment. Inparticular, a kit for determining the presence of antigens in a testsample comprises a) an antibody as defined herein or portion thereof;and b) a conjugate comprising a second antibody (having specificity forthe antigen) attached to a signal generating compound capable ofgenerating a detectable signal. The kit may also contain a control orcalibrator which comprises a reagent which binds to the antigen as wellas an instruction sheet detailing how the kit is to be utilized and thecomponents of the kit.

The present invention also includes a kit for detecting antibodies in atest sample. The kit may comprise a) an anti-antibody specific (forexample, one of the subject invention) for the antibody of interest, andb) an antigen or portion thereof as defined above. A control orcalibrator comprising a reagent which binds to the antigen may also beincluded. More specifically, the kit may comprise a) an anti-antibody(such as the one of the present invention) specific for the antibody andb) a conjugate comprising an antigen (e.g., globulomer) attached to asignal generating compound capable of generating a detectable signal.Again, the kit may also comprise a control of calibrator comprising areagent which binds to the antigen and may also comprise an instructionsheet or package insert describing how the kit should be used and thecomponents of the kit.

The kit may also comprise one container such as vial, bottles or strip,with each container with a pre-set solid phase, and other containerscontaining the respective conjugates. These kits may also contain vialsor containers of other reagents needed for performing the assay, such aswashing, processing and indicator reagents.

It should also be noted that the subject invention not only includes thefull length antibodies described above but also portions or fragmentsthereof, for example, the Fab portion thereof. Additionally, the subjectinvention encompasses any antibody having the same properties of thepresent antibodies in terms of, for example, binding specificity,structure, etc,

Deposit Information: The hybridoma (ML5-8F5.1F2.2A2) which producesmonoclonal antibody 8F5 was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110 on Dec. 1,2005 under the terms of the Budapest Treaty and was assigned ATCC No.PTA-7238.

Hybridoma (ML5-8C5.2C1.8E6.2D5) which produces monoclonal antibody 8C5was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110 on Feb. 28, 2006 under theterms of the Budapest Treaty and was assigned ATCC No. PTA-7407.

The present invention may be illustrated by use of the followingnon-limiting examples:

EXAMPLE 1(a) Production Of Monoclonal Antibodies 8F5 and 8C5

Balb/c mice were immunized sub-q with 50 microgram of A-beta(1-42)globulomer as described in Barghorn et al., 2005, Neurochem, 95,834-847 in CFA (Sigma) and boosted twice at one month intervals. Spleenswere collected and spleen cells fused with mouse myeloma SP2/0 cells at5:1 ratio by a PEG procedure. Fusion cells were plated in 96-well dishesin Azaserine/Hypoxanthine selection media at 2×10⁵ cells/ml, 200 ml perwell. Cells were allowed to grow to form visible colonies andsupernatants assayed for A-beta oligomer reactivity by a direct ELISAassay. Hybridomas secreting antibodies to A-beta oligomers weresubcloned by limiting dilution, until antibody expression appearedstable.

EXAMPLE II 8F5 AND 8C5 Preferential Globulomer Binding Compared toMonomer Preparations of AB(1-40) and AB(1-42)

To test the selectivity of 8F5, two differently dissolved Aβ(1-42)monomer preparations were used as well as freshly prepared Aβ(1-40) assurrogates for monomers. Two types of experiments were performed. In afirst experiment, 8F5 was tested for AS globulomer selectivity by aSandwich-ELISA with globulomer derived but conformer non-specific MAb6G1 (see S. Barghorn et al. J. Neurochemistry, 95:834 (2005)) as acapture antibody, Biotinylated SF5 was used as the second and conformerselective antibody. This experiment is described in Example 2.1 below.

In a second example, described in Example 2,2 below, the oligomerselectivity versus Aβ(1-42) monomer and Aβ(1-40) monomer was examined bydot blot immunoassay. In this experiment, 8F5 exhibited preferentialbinding to Aβ(1-42) globulomer (compared to a known antibody 4G8 mappingto a similar region as 8F5, but derived from immunization with a linearpeptide Aβ(17-24) (Abcam Ltd., Cambridge, Mass.)), as compared toAβ(1-42) monomer as well as compared to Aβ(1-40) monomer. 8C5 was testedin an identical protocol to 8F5.

EXAMPLE 2.1 Oligomer Selectivity Of Monoclonal Antibody 8F5 and 8C5 a)Preparation of Aβ(1-42) Globulomer:

9 mg Aβ(1-42) Fa, Bachem were dissolved in 1.5 ml HFIP (1.1.1.3.3.3Hexafluor-2-propanol) and incubated 1.5 h at 37° C. The solution wasevaporated in a SpeedVac and suspended in 396 μl DMSO (5 mM Aβ stocksolution). The sample was sonified for 20 seconds in a sonic water bath,shaken for 10 minutes and stored over night at −20° C.

The sample was diluted with 4.5 ml PBS (20 mM NaH2PO4; 140 mM NaCl; pH7.4) and 0.5 ml 2% aqueous SDS-solution were added (0.2% SDS content).The mixture was incubated for 7 h at 37° C., diluted with 16 ml H₂O andfurther incubated for 16 hours at 37 deg C. After that, the Aβ(1-42)globulomer solution was centrifuged for 20 min at 3000 g. Thesupernatant was concentrated to 0.5 ml by 30 KDa centriprep. Theconcentrate was dialysed against 5 mM NaH2PO4; 35 mM NaCl; pH7.4overnight at 6° C. Subsequently, the Aβ(1-42) globulomer concentrate wascentrifuged for 10 min at 10000 g. The supernatant was then aliquotedand stored at −20° C.

b) Preparation of Monomer Aβ(1-42), HFIP Pretreated:

3 mg human Aβ(1-42),(Bachem Inc) cat. no. H-1368 were dissolved in 0.5ml HFIP (6 mg/ml suspension) in an 1.7 ml Eppendorff tube and was shaken(Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37° C. till a clearsolution was obtained. The sample was dried in a speed vac concentrator(1.5 h) and resuspended in 13.2 μl DMSO, shook for 10 sec., followed byultrasound bath sonification (20 sec) and shaking (e.g. in EppendorffThermo mixer, 1400 rpm) for 10 min.

6 ml 20 mM NaH2PO4; 140 mM NaCl; 0.1% Pluronic F68; pH 7.4 was added andstirred for 1 h at room temperature. The sample was centrifuged for 20min at 3000 g. The supernatant was discarded and the precipitate solvedin 0.6 ml 20 mM NaH2PO4; 140 mM NaCl; 1% Pluronic F68; pH 7.4. 3.4 mlwater was added and stirred for 1 h at room temperature followed by 20min centrifugation at 3000 g. 8×0.5 ml aliquots of the supernatant werestored at −20°.

c) Preparation of Monomer Aβ(1-42) in NH₄OH:

1 mg Aβ(1-42) solid powder (Bachem Inc. cat. no. H-1368) was dissolvedin 0.5 ml 0.1% NH₄OH in water (freshly prepared) (2 mg/ml) andimmediately shaken for 30 sec. at room temperature to get a clearsolution. The sample was stored at −20° C. for further use

d) Preparation of Monomer Aβ(1-40):

1 mg human Aβ(1-40), (Sachem Inc) cat. no. H-1194 was suspended in 0.25ml HFIP (4 mg/ml suspension) in an Eppendorff tube. The tube was shaken(e.g,, in an Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37° C. toget a clear solution and afterwards dried in a speed vacconcentrator(1.5 h). The sample was redissolved in 46 μl DMSO (21.7mg/mi solution), shaken for 10 sec., followed by 20 sec, sonification inultrasound bath. After 10 min of shaking (e.g. in Eppendorff Thermomixer, 1400 rpm), the sample was stored at −20° C. for further use.

e) Biotinylation of Anti-Aβ Mouse Mab 8F5:

500 μl anti-Aβ mouse Mab 8F5 (0.64 mg/ml) in PBS were added to 2 μl 20mg/ml Sulfo-NHS-Biotin (Pierce Inc. cat. no. 21420) freshly dissolved inwater and shaken (e.g. in Eppendorff Thermo mixer, 1400 rpm), for 30min, dialyzed 16 h at 6° C. in a dialysis tube against 500 ml 20 mM NaPi; 140 mM NaCl; pH 7.4. The dialysate was stored at −20° C. for furtheruse. 8C5 was biotinylated accordingly.

f) Sandwich-ELISA for Aβ-samples:

g) Reagent List:

-   1. F96 Cert. Maxisorp NUNC-Immuno Plate Cat.No.: 439454-   2. Binding antibody:    -   Anti-Aβ mouse MAb 6G1, solved in PBS; conc.: 0.4 mg/ml; store at        −20° C.-   3. Coating-Buffer:    -   100 mM Sodiumhydrogencarbonate; pH 9.6-   4. Blocking Reagent for ELISA; Roche Diagnostics GmbH Cat. No.:    1112589-   5. PBST-Buffer:    -   20 mM NaH2PO4; 140 mM NaCl; 0.05% Tween 20; pH 7.4-   6. Albumin bovine fraction V, protease-free; Serva Cat. No.:    11926.0.3; store at 4° C.-   7. PBST+0.5% BSA-Buffer:    -   20 mM NaH2PO4; 140 mM NaCl; 0.05% Tween 20; pH 7.4+0.5% BSA-   8. Aβ(1-42)-globulomer Standard Stock:    -   solution in 5 mM NaH2PO4; 35 mM NaCl; pH7.4; conc.: 10.77 mg/ml;        store at −20° C.-   9. Aβ(1-42) monomer HFIP treated Standard Stock:    -   solution in 3 mM NaH2PO4; 21 mM NaCl; 0.15% Pluronic F68; pH        7.4; conc.: 0.45 mg/ml; store at −20° C.-   10. Aβ(1-42) monomer in NH4OH Standard Stock; solution in 0.1% NH₄OH    conc.: 2 mg/ml; store at −20° C.-   11. Aβ(1-40) monomer HFIP treated Standard Stock;    -   solution in DMSO; conc.: 21.7 mg/ml; store at −20° C.-   12. biotinylated anti-Aβ mouse mAb clone 8F5; solution in PBS;    conc.: 0.24 mg/ml; store at −80° C.-   13. Streptavidin-POD conjugate; Fa.Roche Cat, No.: 1089153-   14. staining:    -   TMB; Roche Diagnostics GmbH Cat.No.: 92817060; 42 mM in DMSO; 3%        H₂O₂ in water; 100 mM sodium acetate pH 4.9-   15. Stop staining by adding 2M Sulfonic Acid solution    Preparation of reagents:

The following protocol was utilized:

-   -   1. Binding antibody    -   Thaw mMAb 6G1 stock solution and dilute 1:400 in coating buffer,    -   2. Blocking reagent:    -   Dissolve blocking reagent in 100 ml water to prepare the        blocking stock solution and store aliquots of 10 ml at −20° C.        Dilute 3 ml blocking stock solution with 27 ml water for each        plate to block..    -   3. Aβ Standard solutions:        -   a) Aβ(1-42)-globulomer            -   Add 1 μl Aβ(1-42)-globulomer standard stock solution to                1076 μl PBST+0.5% BSA=10 μg/ml            -   Add 50 μl 10 μg/ml Aβ(1-42)-globulomer standard solution                to 4950 μl PBST+0.5% BSA=100 ng/ml        -   b) Aβ(1-42) monomer HFIP-treated            -   Add 10 μl Aβ(1-42) monomer HFIP-pretreated standard                stock solution to 440 μl PBST+0.5% BSA=10 μg/ml            -   Add 50 μl 10 μg/ml Aβ(1-42) monomer HFIP pretreated                standard solution to 4950 μl PBST+0.5% BSA=100 ng/ml        -   c) Aβ(1-42) monomer in NH4OH            -   Add 5 μl Aβ(1-42) monomer in NH4OH standard stock                solution to 995 μl PBST+0.5% BSA=10 μg/ml            -   Add 50 μl 10 μg/ml Aβ(1-42) monomer in NH4OH standard                solution to 4950 μl PEST+0.5% BSA=100 ng/ml        -   d) Aβ(1-40) monomer HFIP-pretreated            -   Add 1 μl Aβ(1-40) monomer HFIP pretreated standard stock                solution to 49 μl PBST+0.5% BSA=430 μg/ml            -   Add 10 μl 430 μg/ml Aβ(1-40) monomer HFIP pretreated                standard solution to 420 μl PBST+05% BSA=10 μg/ml            -   Add 50 μl 10 μg/ml Aβ(1-40) monomer HFIP pretreated                standard solution to 4950 μl PBST+0.5% BSA=100 ng/ml                Standard curves:

No PBST + Final Conc. Stock 0.5% BSA 1 2 ml S 0 ml  100 ng/ml 2 0.633 ml(1) 1.367 ml 31.6 ng/ml 3 0.633 ml (2) 1.367 ml   10 ng/ml 4 0.633 ml(3) 1.367 ml 3.16 ng/ml 5 0.633 ml (4) 1.367 ml   1 ng/ml 6 0.633 ml (5)1.367 ml 0.32 ng/ml 7 0.633 ml (6) 1.367 ml  0.1 ng/ml 8 0 ml 2 ml  0.0ng/ml

-   -   1. Primary antibody: biotinylated mMAb 8F5:    -   The concentrated biotinylated anti-AS mAb 8F5 was diluted in        PBST+0.5% BSA-buffer. The dilution factor was 1/1200=0.2 μg/ml.        The antibody was used immediately.    -   2. Label Reagent:    -   Reconstitute Streptavidin-POD conjugate lyophilizate in 0.5 ml        water. Add 500 μl glycerol and store aliquots of 100 μl at        −20° C. for further use.    -   Dilute the concentrated label reagent in PBST-Buffer.    -   The dilution factor is 1/10000. Use immediately.    -   3. Staining Solution TMB:    -   Mix 20 ml 100 mM sodium acetate pH 4.9 with 200 μl of the    -   TMB solution and 29.5 μl 3% peroxide solution. Use immediately.

Sample Plate Setup: (Note that all standards are run in duplicate)

1 2 3 4 5 6 7 8 9 10 11 12 A 100 100 100 100 100 100 100 100 100 100 100100 B 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 C 1010 10 10 10 10 10 10 10 10 10 10 D 3.16 3.16 3.16 3.16 3.16 3.16 3.163.16 3.16 3.16 3.16 3.16 E 1 1 1 1 1 1 1 1 1 1 1 1 F 0.32 0.32 0.32 0.320.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 G 0.1 0.1 0.1 0.1 0.1 0.1 0.10.1 0.1 0.1 0.1 0.1 H 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Procedure Utilized:

-   1. Apply 100 μl anti-Ag mMAb 6G1 solution per well and incubate    overnight at 4° C.-   2. Discard the antibody solution and wash the wells with 250 μl    PBST-Buffer three times.-   3. Add 260 μl block solution per well and incubate 2 h at room    temperature.-   4. Discard the block solution and wash the wells with 250 μl    PBST-Buffer three times.-   5. After preparation of standards, apply 100 μl per well of    standards to the plate. Incubate 2 h at room temperature and    overnight at 4° C.-   6. Discard the standard solution and wash the wells with 250 μl    PEST-Buffer three times.-   7. Add 200 μl primary biotinylated antibody 8F5 solution per well    and incubate 1.5 h at room temperature.-   8. Discard the antibody solution and wash the wells with 250 μl    PBST-Buffer three times.-   9. Add 200 μl label solution per well and incubate 1 h at room    temperature.-   10. Discard the label solution and wash the wells with 250 μl    PBST-Buffer three times.-   11. Add 100 μl of TMB solution to each well and incubate at room    temperature (5-15 min).-   12. Observe staining and apply 50 μl of the Stop Solution per well    after beginning of background staining.-   13. UV-read at 450 nm.-   14. Calculate results from standard curve.-   15. Evaluation

The results are shown in FIG. 1 for the antibody BF5 and in

FIG. 8 for the antibody 8C5. Log EC50 values are significantly lowestfor the Aβ(1-42) globulomer antigen (1.958) compared to reduced valuesfor two differently prepared Aβ(1-42) monomers (2.745 and 3.003respectively) and Aβ(1-40)monomer (2,825). These data indicate about 10fold selectivity of antibody 8F5 for Aβ(1-42) globulomer versus Aβ(1-42)monomer.

Almost identical results were obtained with antibody 8C5 and are shownin FIG. 8.

EXAMPLE 2.2 Oligomer Selectivity of Monoclonal Antibody 8F5 and 8C5

Discrimination of Aβ monomer against Aβ globulomer by dot blot method:Comparison of 8F5 and 8C5 versus 4G8.

Serial dilutions of Aβ(1-42) globulomer, Aβ1-42 monomer and 41-40monomer were made in the range from 100 pmol/μl-0.01 pmol/μl in PBS. Ofeach sample, 1 μl was dotted onto a nitrocellulose membrane. The mousemonoclonal antibodies 4G8 and 8F5 (0.2 μg/ml) were used for detectionwith an anti-mouse IgG coupled to alkaline phosphatase as secondaryantibody and the staining reagent NBT/BLIP (Roche Diagnostics,Mannheim). The detection signal was analyzed in its intensity(reflective density=RD) via a densitometer (GS 800, Biorad, Hercules,Calif., USA) at an antigen concentration of 10 μmol, At thisconcentration for every AP-form, the measured reflective density was inthe linear range of the densitometer detection. The other antibody 8C5was used in an analogous protocol. The results are shown in Table 1below:

TABLE 1 Discrimination of anti-Aβ-antibodies of Aβ1-40 monomer andAβ1-42 monomer. The discrimination was calculated as the ratio ofdetection signal of Aβ1-42 globulomer and Aβ1-42 monomer, respectivelyAβ1-40 monomer. Reflective Density (RD) [10 pmol] Ratio Ratio Aβ Aβ RDAβ(1-42) RD Aβ(1-42) Aβ (1-42) (1-40) globulomer/ globulomer/ (1-42)mono- mono- RD Aβ(1-42) RD Aβ(1-40) globulomer mer mer monomer monomer8F5 1.6 1.1 0.1 1.4 16.9 8C5 1.3 0.2 0.3 5.1 4.1 4G8 3 3.1 0.7 1 4.2

In particular, the above results indicate that 8F5 and 805 show adifferent binding profile compared to commercially availableanti-Aβ(1-42) antibody to 4G8, which maps to Aβ(17-24) (i.e., a linearsequence). More specifically, 8F5 and 805 show a preference forglobulomer binding versus Aβ42 monomer (see column 4; compare 1.4versus 1) as well as a preference for globulomer binding versus A1340(column 5; compare 16.9 versus 4.2). These two improved bindingselectivities over standard 4GB should result in the production of fewerside effects upon use of BF5 and/or 805, as described above (e.g.,plaque binding).

EXAMPLE III Binding of 8F5 and 8C5 to Aβ(1-42) Fibrils

Since SF5 antibody was generated against soluble globulomers, it washypothesized that 8F5 should not bind to deposited plaque or fibrilmaterial. Therefore, binding of 8F5 to polymerized Aβ fibril suspensionswas tested as described in the following example:

Preparation of Aβ(1-42) Fibrils:

1 mg Aβ(1-42) (Bachem Inc., Catalog Nr.: H-1368) was dissolved in 500 μlaqueous 0.1% NH₄OH (Eppendorff tube), and the sample was stirred for 1min at room temperature followed by 5 min centrifugation at 10000 g.Supernatant was pipetted into a new Eppendorff tube and the Aβ(1-42)concentration measured according to Bradford protein concentration assay(BIO-RAD Inc. assay procedure).

100 μl of this freshly prepared Aβ(1-42) solution were neutralized with300 μl 20 mM NaH2PO4; 140 mM NaCl; pH 7.4 followed by 2% HCl to adjustpH 7.4. The sample was incubated for another 20 hrs at 37° C. andcentrifuged (10 min, 10000 g).

The supernatant was discarded and the fibril pellet resuspended with 400μl 20 mM NaH2PO4; 140 mM NaCl; pH 7.4 under 1 min stirring on a Vortexmixer followed by centrifugation (10 min, 10000 g). After discarding thesupernatant, this resuspending procedure was repeated, and the finalfibril suspension spun down by another centrifugation (10min, 10000 g).The supernatant was once again discarded and the final pelletresuspended in 380 μl 20 mM NaH2PO4; 140 mM NaCl; pH7.4 under 1 minstirring on a Vortex mixer. Aliquots of the sample were stored at −20°C. in a freezer.

80 μl fibril suspension were mixed with 320 μl 20 mM NaH2PO4; 140 mMNaCl; 0.05% Tween 20; pH 7.4, buffer and stirred for 5 min at roomtemperature followed by sonification (20 sec). After centrifugation (10min, 10000 g), the pellet was resuspended with 190 μl 20 mM NaH2PO4; 140mM NaCl; 0.05% Tween 20; pH 7.4 under stirring in a Vortex mixer.

Binding of Antibodies to Aβ(1-42) fibrils

10 μl aliquots of this fibril suspension was incubated with:

-   a) 10 μl 20 mM Na Pi; 140 mM NaCl; pH 7.4-   b) 10 μl 0.1 μg/μl mMAb 6E10 Signet Inc. Cat. #9320 in 20 mM-   c) NaH2PO4; 140 mM NaCl; pH 7.4-   d) 10 μl 0.1 μg/μl mMAb 4G8 Signetlnc. Cat #9220 in 20 mM Na Pi; 140    mM NaCl; pH 7.4-   e) 10 μl 0.1 μg/μl mMAb 8F5 (8C5) in 20 mM Na Pi; 140 mM NaCl; pH    7.4

Samples were incubated for 20 h at 37° C. Finally the samples werecentrifuged (10 min at 10000 g). The supernatants containing the unboundantibody fraction were collected and mixed with 20 μl SDS-PAGE samplebuffer. The pellet fractions were washed with 50 μl 20 mM NaH2PO4; 140mM NaCl; pH 7.4 buffer under 1 min stirring in a Vortex mixer followedby centrifugation (10 min, 10000 g). The final pellets were resuspendedin 20 μl 20 mM Na Pi; 140 mM NaCl; 0.025% Tween 20; pH 7.4 buffer andsolved in 20 μl SDS-PAGE buffer.

SDS-PAGE Analysis

Supernatants and resuspended pellet samples were heated for 5 min at 98°C. and loaded onto a 4-20% Tris/Glycin Gel under the followingconditions:

SDS-sample buffer: 0.3 g SDS; 0.77 g DTT; 4 ml 1M Tris/HCl pH 6.8; 8 mlglycerol; 1 ml 1% Bromphenolblue in Ethanol; add water to 50 ml 4-20%Tris/Glycin Gel:Invitrogen Inc., No.: EC6025BOX

running buffer: 7.5 g Tris; 36g Glycine; 2.5 g SDS; add water to 2.5 l

The PAGE was run at 20 mA. Gels were stained by Coomassie Blue 8250.

Results:

Coomassie staining of SDS-PAGE indicated the presence of heavy and lightchains of antibodies predominantly in the supernatant of the fibrilsuspension (lane 7, FIG. 2), the remaining fibril suspension showed verylittle antibody material while also showing partly depolymerized Abetaat 4.5 kDa. In contrast to 8F5 and 8C5, other anti-Aβ antibodies did notshow up in the soluble fraction (6E10, lane 3, FIG. 2) or only partly(4G8, lane 5, FIG. 2) compared to fibril bound fraction (lane 6, FIG.2).

The relative binding to fibril type Abeta was evaluated from SDS-PAGEanalysis by measuring the Reflective Density values from the heavy chainof the antibodies in the fibril bound and the supernatant fractions andcalculated according to the following formula:

Fibril bound Abfraction=RD_(fibril faction)×100%/(RD_(fibril faction)+RD_(supernatant fraction))

The following values were obtained:

antibody Fibril bound Ab fraction 6E10 98% 8F5 16% 8C5 21%

These data indicate a significant reduction of bound 8F5 and 8C5compared to standard antibody 6E10.

EXAMPLE IV Preferential Binding of Endogenous Aβ(1-42) GlobulomersCompared to Aβ(1-40)

Based upon the oligomer concept of Aβ, it is important that anti-Aβoligomer antibodies also can demonstrate preferential binding forAβ(1-42) oligomers in vivo, in particular, over Aβ(1-40) in MildCognitive Impairment and AD patients. The concept of lowering Aβ(1-42)species over Aβ(1-40) is used in a therapeutic approach for thetreatment of AD via NSAIDs (Weggen et al., Nature 414, 212-216 (2001)).It is assumed that those NSAIDs which lower Aβ(1-42) in relation toAβ(1-40) display the best efficacy in the treatment of AlzheimerDisease. The Aβ(1-42)/Aβ(1-40)ratio is important for a selective therapyas well as for diagnostic purposes.

An analysis was performed with CSF samples from Alzheimer's Diseasepatients and patients with MCI. From the results shown in FIG. 3 anddescribed below, it can be concluded that 8F5 has a major advantage overAβ antibodies like 6E10 because 8F5 detects a higher ratio of Aβ(1-42)over less aggregating Aβ(1-40). This advantage will allow one to moreselectively diagnose and neutralize Aβ(1-42) type oligomers in MCI andAD patients.

A) Endogenous Amyloid β(1-42) and Amyloid β(1-40) Levels in CSF of MCIand AD Patients after Immunoprecipitation with Oligomer SelectiveAnti-Aβ Murine Mab 8F5:

Immobilization of anti-Aβ mMAB's to CNBr-activated Sepharose 4B:

-   -   a) mMAb 6E10 Signet Inc., Cat.no. 9320    -   b) mMAb 8F5

0.4 g CNBr-activated Sepharose 4B (Amersham Pharmacia Bio-tech AB,Uppsala, Sweden, Inc., No.: 17-0430-01) were added to 10 ml aqueous 1 mMHCl and incubated for 30 min at room temperature. The CNBr-activatedSepharose 4B was washed three times with 10 ml 1 mM HCl and twice with10 ml 100 mM NaHCO₃; 500 mM NaCl; pH 8.3. For each of the immobilizedantibodies, 100 μl CNBr-activated Sepharose 4B Matrix were added to 950μl 0.5 mg/ml anti-Aβ mMAb solution in 100 mM NaHCO₃; 500 mM NaCl; pH8.3. After 2 h of shaking at room temperature, samples were centrifugedfor 5 min at 10000 g. Then, 500 μl 100 mM Ethanolamine; 100 mM NaHCO₃;500 mM NaCl; pH 8.3, buffer was added to the beads, and samples wereshaken for lh at room temperature. The anti-AS mMAb-Sepharose sampleswere centrifuged for 5 min at 10000 g and washed 5 times with 500 μl 20mM NaH2PO4; 140 mM NaCl; pH 7.4. Before storage at 6° C., samples werestabilized by adding sodium azide to 0.02% final concentration.

Immunoprecipitation:

-   -   a) mMAb 6E10-Sepharose    -   b) mMAb 8F5 -Sepharose

200 μl of the human Cerebral Spinal Fluid samples were diluted with 200μl 20 mM NaH2PO4NaH₂PO₄; 140 mM NaCl; 0.05% Tween 20; pH 7.4. Thesesamples were added to 2 μl anti-Ai mMAb-Sepharose Matrix and stirred for2 h at room temperature. The samples were centrifuged for 5 min at 10000g. The supernatants were discarded and the anti-AS mMAb-Sepharose washedtwice with 50 μl PBS, stirred for lmin and centrifuged (5 min at 10000g). The supernatants were discarded, and the Sepharose beads were nowsuspended in 50 μl 2 mM NaH₂PO₄NaH2PO4; 14 mM NaCl, pH7.4, followed bylmin of stirring at room temperature and 5 min of centrifugation at10000 g. In a next step, the anti-Aβ mMAb-Sepharose beads were treatedwith 50 μl 50% CH₃CN; 0.2% TFA in water. After 10 min shaking at roomtemperature, samples were centrifuged 5 min at 10000 g. The supernatantswere collected and transferred to 1.5 ml Eppendorf tubes. Samples weremixed with 50 μl water and evaporated in a Speed Vac concentrator.

The pellet was redissolved in 4 μl 70% HCOOH, shaken for 10 min at roomtemperature and neutralized with 76 μl 1M Tris-solution and 720 μl 20 mMNaH₂PO₄NaH2PO4; 140 mM NaCl; 0.05% Tween 20; pH 7.4.

Samples for the Determination of Aβ(1-40); (1-42) Monomer Forms in CSF:

-   a) Aβ-content in CSF-samples without immunoprecipitation:    -   158 μl CSF were diluted with 342 μl 20 mM NaH₂PO₄; 140 mM NaCl;        0.05% Tween 20; pH 7.4. This 1:3.16 dilution was taken for        Sandwich ELISA's and taken into account during evaluation.-   b) Ag-content in CSF-samples after immunoprecipitation:    -   Samples from the above-mentioned procedure were taken for        analysis.        Sandwich-ELISA Protocol used for the Determination of Aβ(1-40)        in CSF

Reagent List:

-   1. F96 Cert. Maxisorp NUNC-Immuno Plate Cat.No.: 439454-   2. Binding antibody    -   Anti-Aβ mAb clone 6E10; Signet Cat. No. 9320; conc.:    -   0.4 mg/ml Bradford (BioRad); store at −20° C.-   3. Coupling-buffer    -   100 mM sodiumhydrogencarbonate; pH9.6-   4. Blocking Reagent for ELISA; Roche Diagnostics GmbH Cat. No.:    1112589-   5. PBST-buffer    -   20 mM NaH₂PO₄NaH2PO4; 140 mM NaCl; 0.05% Tween 20; pH7.4-   6.Aβ(1-40) Standard:    -   Aβ(1-40) solid powder; Bachem Cat. No.: H-1194; store at −20° C.-   7. Primary antibody:    -   anti-Aβ(1-40) rabbit pAb; affinity purified; solution in PBS;        conc.: 0.039 mg/ml;        -   Signet Cat. No. 9130-005; store at −20° C.-   8. Label reagent:    -   anti-rabbit-POD conjugate; Fa.Jackson ImmunoResearch Cat.No.:        111-036-045;-   9. Staining:    -   TMB; Roche Diagnostics GmbH Cat. No.: 92817060; 42 mM in DMSO;        3% H₂O₂ in water; 100 mM sodium acetate pH 4.9-   10. Stop Solution 2M Sulfonic Acid    Protocols used for Preparation of Reagents:-   1. Binding antibody:    -   anti-AS mAb 6E10 (Signet Inc, Catalog #9320) is diluted to a        final concentration of 0.7 microg/ml.-   2. Blocking reagent:    -   For preparation of the blocking stock solution the blocking        reagent is dissolved in 100 ml H₂O and stored at −20° C. in        aliquots of 10 ml each. 3 ml of the blocking stock solution are        diluted with 27 ml H₂O for blocking one ELISA plate.-   3.Aβ(1-40) monomer form standard dilution:    -   A) Aβ(1-40) monomer Standard Stock: dissolve 0.5 mg Aβ(1-40) in        250 μl 0.1% NH₄OH, conc.: 2 mg/ml; freshly prepared; use        immediately,    -   B) Add 5 μl Aβ(1-40)-monomer standard stock solution to 995 μl        PBST=10 μg/ml    -   C) Add 5 μl, 10 μg/ml Aβ(1-40)-monomer standard solution to 4995        μl PBST=10 ng/ml

Standard Curve:

No Final conc. Stock PBST 1 2 ml B 0 ml 10000 pg/ml  2 0.633 ml (1)1.367 ml 3160 pg/ml  3 0.633 ml (2) 1.367 ml 1000 pg/ml  4 0.633 ml (3)1.367 ml 316 pg/ml 5 0.633 ml (4) 1.367 ml 100 pg/ml 6 0.633 ml (5)1.367 ml 31.6 pg/ml  7 0.633 ml (6) 1.367 ml  10 pg/ml 8 0 ml 2 ml  0.0pg/ml

Samples:

IP: immunoprecipitate samples No dilution factor sample PBST 1 0.4 ml IP  0 ml directly 2 0.1 ml (1) 0.4 ml 1:5 3 0.1 ml (2) 0.4 ml 1:25 4 0.1ml (3) 0.4 ml 1:125

-   4. Primary antibody:    -   Dilute the concentrated anti-Aβ(1-40) pAb in PBST buffer. The        dilution factor is 1/200=0.2 μg/ml. Use immediately.-   5. Secondary antibody:    -   Lyophilized anti-rabbit-POD conjugate is dissolved in 0.5 ml H₂O        and mixed with 500 μl glycerol. The antibody concentrate is then        stored at −20° C. in aliquots of 100 μl. The concentrate is        diluted 1:10′000 in PBST buffer. The antibody solution is used        immediately.-   6.TMB solution:    -   20 ml of 100 mM sodium acetate, pH 4.9, are mixed with 200 μl        TMB solution and 29.5 μl of 3% hydrogen peroxide. This solution        is used immediately.        Sample Plate Setup: (Note that all standards and samples are run        in duplicate.)

1 2 3 4 5 6 7 8 9 10 11 12 A 10000 10000 U1 U1 B 3160 3160 U2 U2 C 10001000 U3 U3 D 316 316 U4 U4 E 100 100 U5 U5 F 31.6 31.6 U6 U6 G 10 10 U7U7 H 0.0 0.0 U8 U8 U1-U# = Unknown samples

Procedure Used:

-   1. Apply 100 μl binding antibody solution per well and incubate    overnight at 4° C.-   2. Discard the antibody solution and wash the wells with 250 μl    PBST-buffer for three times.-   3. Add 260 μl block solution per well and incubate 2 h at room    temperature.-   4. Discard the block solution and wash the wells with 250 μl    PBST-buffer for three times.-   5. After preparation of standards and samples, apply 100 μl per well    of standards and samples to the plate and incubate 2 h at room    temperature and overnight at 4° C.-   6. Discard the standard/sample solution and wash the wells with 250    μl PBST-buffer for three times.-   7. Add 200 μl primary antibody solution per well and incubate 1.5 h    at room temperature.-   8. Discard the antibody solution and wash the wells with 250 μl    PEST-buffer for three times.-   9. Add 200 μl label solution per well and incubate 1 h at room    temperature.-   10. Discard the label solution and wash the wells with 250    μPEST-buffer for three times.-   11. Add 100 μl of TMB solution to each well and incubate at room    temperature (5-15 min).-   12. Observe colour development and apply 50 μl of the Stop solution    per well.-   13. Read at 450 nm.-   14. Calculate results from standard curve.-   15. Evaluation:

If extinction from unknown samples is not in the linearity range of thecalibration curve, repeat ELISA with appropriated sample dilution.

Sandwich-ELISA Protocol Used for the Determination of Aβ(1-42) MonomerForm in CSF Reagent List:

-   1, F96 Cert. Maxisorp NUNC-Immuno Plate Cat.No.:439454-   2. Binding antibody    -   Anti-AE rnAb clone 6E10; Signet Cat.No. 9320; conc.:

0.4 mg/ml Bradford (BioRad); store at −20° C.

-   3. Coating-Buffer    -   100 mM sodiumhydrogencarbonate; pH9.6-   4. Blocking Reagent for ELISA; Roche Diagnostics GmbH Cat.No.:    1112589-   5. PBST-Buffer    -   20 mM NaH2PO4NaH2PO4; 140 mM NaCl; 0.05% Tween 20; pH7.4-   6.Aβ(1-42) Standard:    -   Aβ(1-42) solid powder; Bachem Cat. No.: H-1368; store at −20° C.-   7. Primary antibody:    -   anti-Aβ(1-42) rabbit pAb; affinity purified; biotinylated;        solution in PBS with 50% glycerol; conc.: 0.25 mg/ml; Signet        Cat.No. 9137-005; store at −20° C.-   8.Label reagent:    -   anti-rabbit-POD conjugate; Fa. Jackson ImmunoResearch Cat. No.:        111-036-045-   9. Staining:    -   TMB; Roche Diagnostics GmbH Cat. No.: 92817060;    -   42 mM in DMSO    -   3% H₂O₂ in water    -   100 mM sodium acetate, pH4.9    -   Stop Solution: 2M Sulfonic Acid

Method Used in Preparation of Reagents:

1. Binding antibody:

Dilute anti-Ag mAb clone 6E10 1:400 in coating buffer.

2. Blocking reagent:

Dissolve blocking reagent in 100 ml water to prepare the blocking stocksolution and store aliquots of 10 ml at −20° C.

Dilute 3 ml blocking stock solution with 27 ml water for each plate toblock.

3. Aβ(1-42) monomer form, standard dilution:

Aβ(1-42) Monomer Standard Stock: dissolve 0.5 mg Aβ(1-42) in 250 μl 0.1%NH₄OH; conc.: 2 mg/ml; freshly prepared; use immediately.

Add 5 μl Aβ(1-42)-monomer standard stock solution to 995 μl PBST=10μg/ml.

Add 5 μl, 10 μg/ml Aβ(1-42)-monomer standard solution to 4995 μl PBST=10ng/ml.

Standard Curve:

No Final conc. Stock PBST 1 2 ml B 0 ml 10000 pg/ml  2 0.633 ml (1)1.367 ml 3160 pg/ml  3 0.633 ml (2) 1.367 ml 1000 pg/ml  4 0.633 ml (3)1.367 ml 316 pg/ml 5 0.633 ml (4) 1.367 ml 100 pg/ml 6 0.633 ml (5)1.367 ml 31.6 pg/ml  7 0.633 ml (6) 1.367 ml  10 pg/ml 8 0 ml 2 ml  0.0pg/ml

Samples:

IP: immunoprecipitate samples No dilution factor sample PBST 1 0.4 ml IP  0 ml directly 2 0.1 ml (1) 0.4 ml 1:5 3 0.1 ml (2) 0.4 ml 1:25 4 0.1ml (3) 0.4 ml 1:125

Procedure Used:

-   1. Primary antibody:    -   Dilute the concentrated anti-Aβ(1-42) pAb in PEST buffer. The        dilution factor is 1/1250=0.2 μg/ml. Use immediately.-   2. Label Reagent:    -   Reconstitute anti-rabbit-POD conjugate lyophilizate in 0.5 ml        water. Add 50 μl glycerol and store aliquots of 100 μl at        −20° C. for further use.    -   Dilute the concentrated Label reagent in PEST-buffer. The        dilution factor is 1/5000. Use immediately.-   3. TMB solution:    -   Mix 20 ml 100 mM sodium acetate pH4.9 with 200 μl of the TMB        solution and 29.5 μl 3% Peroxide solution. Use immediately.        Sample Plate Setup: (Note that all standards and samples are run        in duplicate.)

1 2 3 4 5 6 7 8 9 10 11 12 A 10000 10000 U1 U1 B 3160 3160 U2 U2 C 10001000 U3 U3 D 316 316 U4 U4 E 100 100 U5 U5 F 31.6 31.6 U6 U6 G 10 10 U7U7 H 0.0 0.0 U8 U8 U1-U# = Unknown samples

Procedure Used:

-   1. Apply 100 μl binding antibody solution per well and incubate    overnight at 4° C.-   2. Discard the antibody solution and wash the wells with 250 μl    PBST-buffer for three times.-   3. Add 260 μl block solution per well and incubate 2 h at room    temperature.-   4. Discard the block solution and wash the wells with 250 μl    PBST-buffer for three times.-   5. After preparation of standards and samples, apply 100 μl per well    of standards and samples to the plate. Incubate 2h at room    temperature and overnight at 4° C.-   6. Discard the standard/sample solution and wash the wells with 250    μl PBST-buffer for three times.-   7. Add 200 μl primary antibody solution per well and incubate 1.5 h    at room temperature.-   8. Discard the antibody solution and wash the wells with 250 μl    PBST-buffer for three times.-   9. Add 200 μl label solution per well and incubate 1 h at room    temperature.-   10. Discard the label solution and wash the wells with 250 μl    PBST-buffer for three times.-   11.Add 100 μl of TMB solution to each well and incubate at room    temperature (5-15 min).-   12. Observe color staining and apply 50 μl of the Stop Solution per    well.-   13. Read at 450 nm,-   14. Calculate results from standard curve.-   15. Evaluation:

If extinction from unknown samples is not in the linearity range of thecalibration curve, repeat ELISA with appropriate sample dilution.

Results:

Aβ40 ELISA (Signet) Aβ42 ELISA (Signet) Aβ(1-40) SEM Aβ(1-42) SEMAβ42/40 MCI samples (n = 4) without IP 11678.9 2879.4 1242.0 353.5 7.84%6E10 IP 8282.4 2185.7 2035.1 280.9 17.35% 8F5 IP 8586.1 2396.8 2654.6411.4 20.95% AD samples (n = 2) without IP 7297.5 1464.5 843.0 157.510.95% 6E10 IP 5610.2 28.3 1453.0 14.5 20.57% 8F5 IP 4133.9 86.9 1670.212.3 28.78%

The above results indicate the following:

-   a. A globulomer preferential antibody like 8F5 (or 8C5), in    comparison to a non-globulomer selective antibody like 6E10, binds    preferentially to Aβ42 compared to Aβ40 independent from the disease    state. This result is indicative of a successful treatment for    Alzheimer's Disease because preferentially eliminating Aβ42 over    Aβ40 is being followed as a concept in AD-treatment, e.g., by the    use of R-flubiprofen, Flurizan which has demonstrated efficacy in AD    treatment in a clinical trial published by Myriad Inc. This concept    was published by S. Weggen et al. (J Biol Chem. (2003)    278(34):31831-7). The results are shown in FIG. 3. b. A globulomer    preferential antibody like 8F5 (or 8C5) binds to even more Aβ42 than    Aβ40 in patients compared to healthy controls. This result is even    more indicative of a successful treatment for Alzheimer's Disease    because, as noted above, preferentially eliminating Aβ42 over Aβ40    is being followed as a concept in AD-treatment (e.g., by the use of    non-steroidal anti-inflammatory drugs, like R-flubiprofen). (See    FIG. 3.)    B) Endogenous Amyloid β(1-42) and Amyloid β(1-40) Levels in Human    CSF After Immunoprecipitation with Globulomer Selective Anti-Aβ    Murine MAB 8F5 or 8C5 in Comparison with Globulomer Unselective    Antibody 6E10:

b1) Immunoprecipitation (IP) with Dynabeads M-280 Sheep anti-Mouse IgG

Abeta-antibody solutions

The following pure antibodies were obtained from hybridomas according tostandard purification procedures:

-   -   mMab 6E10; Fa.Signet. Nr.: 9320; 1 mg/ml in PBS buffer    -   mMab 8F5; 1.65 mg/ml in PBS buffer    -   mMab 805; 1.44 mg/ml in PBS buffer

Dynabeads M-280 Sheep anti-Mouse IgG:

Sheep anti-Mouse IgG (Invitrogen Inc., Cat. no.: 112.02) is covalentlybound to magnetic beads (Dynabeads).

Activation of Dynabeads with monoclonal mouse antibodies

-   -   The stock-suspension of dynabeads (Dynabeads M-280 Sheep        anti-Mouse IgG, Invitrogen; Prod. No. 112.02) was shaken        carefully to prevent foaming.    -   1 mL was aseptically removed and transferred to a 1.5 mL        reaction vial.    -   The dynabeads were washed 3 times 5 min with 1 mL        immunoprecipitation (IP)-wash buffer (IP-wash-buffer: PBS (20 mM        NaH₂PO₄, 140 mM NaCl, pH 7.4), 0.1% (w/v) BSA). During the        washing procedure, the supernatant was carefully removed while        the dynabeads were immobilized at the side of the reaction vial        with a magnetic separator stand (MSS).    -   The washed dynabeads were incubated with 40 μg Abeta-antibody in        1 mL PBS, 0.1% (w/v) BSA.    -   The activation was carried out by overnight incubation under        shaking at 4° C.    -   The activated dynabeads were washed 4 times 30 min (again using        the MSS) with 1 mL IP-wash buffer (PBS (20 mM NaH₂PO₄, 140 mM        NaCl, pH 7.4), 0.1% (w/v) BSA).    -   The activated dynabeads were resuspended with 1 mL PBS, 0.1%        (w/v) BSA, 0.02 (w/v)% Na-Azide; vortexed and centrifuged        briefly.    -   The antibody activated dynabeads were stored at 4° C. until        further use.

CSF Sample Preparation:

400 μL CSF from an Alzheimer's disease patient were added to 4 μLComplete Protease Inhibitor Cocktail (Roche Inc. Cat. no.: 1697498, 1tablet dissolved in 1 mL water) and 0.8 μL 500 mM PMSF dissolved inmethanol. After 10 min., 1.6 mL 20 mM NaH₂PO₄, 140 mM NaCl, 0.05% Tween20, pH 7.4 (PBST) was added.

Immunoprecipitation of Abeta Species from Human AD-CSF:

250 μL aliquot of the prepared CSF sample were added to 25 μLanti-Aβ-Dynabeads suspension.

-   -   Immunoprecipitation occurred under stirring at 6° C. for 16        hours. Subsequent washing of the beads was performed 3 times 5        min. with 1 mL PBS/0.1% (w/v) BSA and finally once 3 min. with 1        mL 10 mM Tris/HCL pH 7.5 buffer. During the washing procedure,        the supernatant was carefully removed while the dynabeads were        immobilized at the side of the reaction vial with a magnetic        separator stand (MSS),

The residual supernatant was thoroughly removed after the final washingstep. The Abeta peptides and the corresponding antibody were removedfrom the Dynabeads by adding 25 μL sample buffer withoutβ-Mercaptoethanol (0.36 M Bistris, 0.16 M Bicine, 1% SDS (w/v), 15%(w/v) sucrose, 0.004% (w/v) Bromphenolblue) to the Eppendorff tube andheating for 5 min at 95° C. in a heating block. After cooling to roomtemperature, the dynabeads were immobilized at the side of the reactionvial with a magnetic separator stand (MSS), and the supernatant wastransferred to another Eppendorff tube (IP eluate).

Analysis of Abeta Immunoprecipitates by Urea-PAGE Followed by WesternBlot Procedure:

The quantification of Aβ1-40 and Aβ1-42 species was performed by a 8 MUrea Poly-Acrylamide-Gel-Electrophoresis system and subsequent WesternBlot analysis according to the procedure first described by H. W. Klafkiet al., Analytical Biochemistry 237, 24-29 (1996) and later also used byJ. Wiltfang et al., J. of Neurochemistry 81, 481-496, 2002. There wereonly two minor changes made in the experimental procedure:

-   -   1) SDS concentration in the stacking gel was adjusted to 0.25%        (w/v) instead of 0.1% (w/v).    -   2) For the Western blot the antibody 1E8 (Senetek Drug Delivery        Technologies Inc. St.Louis, Mo., USA) was replaced by Anti-Human        Amyloid β(N) (82E1) Mouse IgG mAb (IBL, Cat. no: 10323)

15 μL IP eluate aliquots of the immunoprecipitated samples were loadedonto the 8 M Urea PAGE. Electrophoresis was performed at 100 V (15 min)and continued at 60 V. The electrophoresis was stopped when the runningfront of the blue sample loading dye was still 0.5 cm away from the endof the gel.

Western Blot Procedure:

Western blot analysis was performed in a Semi Dry Blotting chamber(BioRad Inc,, 45 min at 75 mA) onto 7.5 cm×9 cm Nitrocellulose 0.45 μm(BioRad Inc.).

Blotting buffer: 6 g Tris; 28.1 g Glycin; 500 mL Methanol; adjust to 2.5l with water.

The Nitrocellulose blot was boiled for 10 min in PBS at 100° C., Theblot was saturated by treatment with 50 mL 5% (w/v) BSA in PBST for 1hour at RT. After removal of the fluid phase, the following washing stepwas performed twice with: 50 mL TTBS (25 mM Tris/HCl; 150 mM NaClPuffer; 0.05% Tween 20; pH 7.5) for 10 min at RT and subsequently with50 mL TBS (25 mM Tris/HCl; 150 mM NaCl buffer; pH 7,5) for 10 min at RT.

For further development, the final washing buffer was discarded from theblot and 15 mL antibody I solution (0.2 μg/mL 82E1=1:500 in 3% (w/v)skimmed milk powder (Lasana Inc.), in 15 mL TES) were added for 20 hoursat 6° C. Removal of buffer was followed by the three wash steps asdescribed above. The blot was incubated with Antibody solution II(1:10000 dilution of anti-Mouse -POD in 15 mL 3% (w/v) skimmed milkpowder in 15 mL TBS) for 1 hour at RT, Removal of buffer was followed bythe three wash steps as described above.

After removal of the last washing buffer, 2 mL Super Signal West FemtoMaximum Sensitivity Substrate Enhancer and 2 mL Peroxide Solution wasmixed. The freshly prepared solution was poured onto the blot which waspreincubated in the dark for 5 min. Chemiluminescence was recorded usinga VersaDoc Imaging system(BioRad).

Imaging parameters:

-   -   exposure time 180 sec.

Picture records after 30 sec., 60 sec., 120 sec. and 180 sec.

The results were obtained from the picture with 180 sec. exposure time.

Aβ40 urea- Aβ42 urea- Ratio Aβ42/ PAGE [pg/ml] PAGE [pg/ml] Aβ40 + 42 ×100% 6E10 IP 4389 202 4.4% 8F5 IP 1260 112 8.1% 8C5 IP 1202 211 14.9%

The above results indicate that a globulomer preferential antibody like8F5 or 8C5, in comparison to a non-globulomer selective antibody like6E10, binds to more Aβ42 than Aβ40 in human CSF. This result isindicative of a successful treatment for Alzheimer's Disease because, asnoted above, preferentially eliminating Aβ42 over Aβ40 is beingfollowing as a concept in AD-treatment (e.g., by the use ofR-flubiprofen (see above)).

EXAMPLE V 8E5 Improves Novel Object Recognition in APP Transgenic Mice

In order to test a positive effect on cognition by neutralizing internalAβ(1-42) globulomer epitope with antibody 8F5, a passive immunizationexperiment with APP transgenic mice was performed in which the mice weretested for their ability to remember objects they have investigatedbefore. After some time, delay between first and second encounter ofobjects, APP transgenic mice are not able to recognize the alreadyinvestigated object. This experiment is based on the natural curiosityof the animals, and a significant lack of interest in the alreadyinvestigated object demonstrates recognition of the object.

EXAMPLE V.1 Increased Recognition Index by Monoclonal Antibody BF5

Animals:

Female mice of a single transgenic mouse model of Alzheimer's Disease inFVB×C57B1 background (APP/L, ReMYND, Leuven, Belgium) and negativelitter mates as wild type controls in FVB×C57B1 background with an ageof 3 months were used. All mice were genotyped by polymerase chainreaction (PCR) at the age of 3 weeks and received a unique identitynumber, once the PCR results were known and were double checked by asecond PCR before the onset of the study. All mice were randomized andage-matched, i.e., they were given a random number by computer andallocated randomly to a treatment. Animals were caged by treatment group18 days before the onset of the study in order to allow them tofamiliarize to the new cage context. Mice had free access topre-filtered and sterile water (UV-lamp) and standard mouse chow. Thefood was stored under dry and cool conditions in a well-ventilatedstorage room. The amount of water and food was checked daily, suppliedwhen necessary and refreshed twice a week. Mice were housed under areversed day-night rhythm: 14 hours light/10 hours darkness starting at7 p.m. in standard metal cages type RVS T2 (area of 540 cm2). The cagesare equipped with solid floors and a layer of bedding litter. The numberof mice per cage was limited in accordance with legislation on animalwelfare. Five days before the onset of the behavior test, mice werereplaced in macrolon Type 2 cages and transported to the laboratory inorder to adapt to the laboratory environment in preparation for thebehavior test.

Treatment (Passive Immunization):

Three individual experiments were performed in which the mice (at least9 per group) received intraperitoneal injections (500 μg in 240μL/mouse) at days 1, 8 and 15. Mice were treated with monoclonalantibodies 6G1, 8F5 and other non-disclosed antibodies, all dissolved inphosphate-buffered saline, or with 320 μL phosphate-buffered saline.

Novel Object Recognition Test:

The novel object recognition test was performed on the day of the thirdtreatment. The protocol used followed the method as described byDewachter et al, (Journal of Neuroscience, 2002, 22(9):3445-3453). Micewere familiarized for one hour to a Plexiglas open-field box (52×52×40cm) with black vertical walls and a translucent floor, dimly illuminatedby a lamp placed underneath the box. The next day, the animals wereplaced in the same box and submitted to a 10 minute acquisition trial.During this trial, mice were placed individually in the open field inthe presence of 2 identical objects A (orange barrel or green cube,similar size of ±4 cm), and the duration (time_(AA)) and the frequency(Freq_(AA)) exploring object A (when the animals snout was directedtowards the object at a distance of <1 cm and the mice were activelysniffing in the direction of the object) was recorded by a computerizedsystem (Ethovision, Noldus information Technology, Wageningen,Netherlands). During a 10 minute retention trial (second trial)performed 2.5 hours later, a novel object (object B, green cube ororange barrel) was placed together with the familiar object (object A)into the open field (Freq_(A) and Freq_(B) and Time_(A) and Time_(B),respectively). The recognition index (RI), defined as the ratio of theduration in which the novel object was explored over the duration inwhich both objects were explored [Time_(B)/(Time_(A)+Time_(B))×100], wasused to measure non-spatial memory. The duration and frequency thatobject A was explored during the acquisition trial (Time_(AA) andFreq_(AA)) was used to measure curiosity.

Analysis of data was done by combining APP transgenic mice that receivedmonoclonal antibodies 6G1 or 8F5 or phosphate-buffered saline, andnon-transgenic littermates that received phosphate-buffered saline, fromall three studies (FIG. 4). Mice that do not distinguish between an oldobject and a novel object have a recognition index of 50. Mice thatrecognize the old object will preferably explore the novel object andhence the recognition index becomes<50. Mice that exclusively explorethe novel object have a recognition index of 100. The mean recognitionindex per group was compared against chance level, i.e., 50, by t-test.The mean recognition index of all groups was also compared by ANOVAfollowed by a post-hoc t-test. The difference between PBS and wild typegroups indicated a cognitive deficit of APP transgenic mice in thisparadigm. PBS-injected mice performed at chance level (i.e., notsignificantly different from 50) while all other mice showed objectrecognition (FIG. 4: stars). When the performance of antibody-treatedAPP transgenic mice was compared with control groups, a significantdifference was found versus PBS-treated but not versus wild-type mice(FIG. 4: circles) indicating that treatment with antibody 8F5 reversedthe cognitive deficit in these APP transgenic mice.

EXAMPLE VI In Situ Analysis of the Specific Reaction of Antibodies 8F5and 8C5 to Fibrillar Amyloid Beta Peptide in the Form of Amyloid Plaquesand Amyloid in Meningeal Vessels in Old APP Transgenic Mice andAlzheimer's Disease Patients

Antibodies 8F5 and 8C5 show reduced staining to fibrillar Aβ peptidedeposits suggesting that their therapeutic effect is mediated by bindingto soluble globulomeric forms rather than fibrillar deposited forms ofAβ peptide. Since antibody binding to fibrillar Aβ peptide can lead tofast dissolution of aggregates and a subsequent increase of soluble Aβconcentration, which in turn is thought to be neurotoxic and could leadto microhemorrhages, an antibody therapy that effects the solubleglobulomer rather than the monomer is preferred.

Methods:

For these experiments, several brain material samples were used:cortical tissue from 2 AD patients (RZ16 and RZ 55)and cortical tissuefrom 19 month old Tg2576 mice (APPSWE # 001349, Taconic, Hudson, N.Y.,USA) or 12 month old APP/L mice (ReMYND, Leuven, Belgium).

The mice overexpress human APP with a familial Alzheimer's diseasemutation and form β-amyloid deposits in the brain parenchyma at about 11months of age and β-amyloid deposits in larger cerebral vessels at about18 months of age. The animals were deeply anaesthetized andtranscardially perfused with 0.1 M phosphate-buffered saline (PBS) toflush the blood. Then, the brain was removed from the cranium anddivided longitudinally. One hemisphere of the brain was shock-frozen andthe other fixated by immersion into 4% paraformaldehyde.

The immersion-fixated hemisphere was cryoprotected by soaking in 30%sucrose in PBS and mounted on a freezing microtome. The entire forebrainwas cut into 40 μm transverse sections which were collected in PBS andused for the subsequent staining procedure. The neocortex samples fromAlzheimer's disease patients were obtained from Brain-Net, Munich,Germany as frozen tissue, immersion-fixated in 4% paraformaldehydeduring thawing, and subsequently treated like the mouse tissue.

Individual sections were stained with Congo Red using the followingprotocol:

Material:

-   -   Amyloid dye Congo Red kit (Sigma-Aldrich; HT-60), consisting of        alcoholic NaCl solution, NaOH solution and Congo Red solution    -   staining cuvettes    -   microscope slides SuperfrostPlus and coverslips    -   Ethanol, Xylol, embedding medium

Reagents:

-   -   NaOH diluted 1:100 with NaCl solution yields alkaline saline    -   alkaline saline diluted 1:100 with Congo Red solution yields        alkaline Congo Red solution (prepare no more than 15 min before        use, filtrate)    -   mount sections on slide and allow them to dry    -   incubate slide in staining cuvette, first for 30-40 minutes in        alkaline saline, then for 30-40 minutes in alkaline Congo Red        solution    -   rinse three times with fresh ethanol and embed over xylol

Staining was first photographed using a Zeiss Axioplan microscope(Zeiss, Jena, Germany) and evaluated qualitatively. Red color indicatedamyloid deposits both in the form of plaques and in larger meningealvessels. Later on, evaluation of antibody staining focused on thesestructures.

Staining was performed by incubating the sections with a solutioncontaining 0.07-0.7 μg/ml of the respective antibody in accordance withthe following protocol:

Materials:

-   -   TBST washing solution (Tris Buffered Saline with Tween 20; 10×        concentrate; DakoCytomation 53306, DAKO, Hamburg, Germany) 1:10        in Aqua bidest.)    -   0.3% H₂O₂ in methanol    -   donkey serum (Serotec, Düsseldorf, Germany), 5% in TBST, as        blocking serum    -   monoclonal mouse-anti-globulomer antibodies diluted at given        concentrations in TBST    -   secondary antibody: biotinylated donkey-anti-mouse antibody        (Jackson Immuno/Dianova, Hamburg, Germany; 715-065-150; diluted        1:500 in TBST)    -   StreptABComplex (DakoCytomation K 0377, DAKO, Hamburg, Germany)    -   Peroxidase Substrate Kit diaminobenzidine (=DAB; SK-4100; Vector        Laboratories, Burlingame, Calif., USA)    -   SuperFrost Plus microscope slides and coverslips    -   xylol free embedding medium (Medite, Burgdorf, Germany; X-tra        Kitt)

Procedure:

-   -   transfer floating sections into ice-cold 0.3% H₂O₂ and incubate        for 30 min    -   wash for 5 min in TBST buffer    -   incubate with donkey serum/TBST for 20 minutes    -   incubate with primary antibody for 24 hours at room temperature    -   wash in TBST buffer for 5 minutes    -   incubate with blocking serum for 20 minutes    -   wash in TBST buffer for 5 minutes    -   incubate with secondary antibody for 60 minutes at ambient        temperature    -   wash in TBST buffer for 5 minutes    -   incubate with StreptABComplex for 60 minutes at ambient        temperature    -   wash in TBST buffer for 5 minutes    -   incubate with DAB for 20 minutes    -   mount the section on slides, air-dry slides, dehydrate slides        with alcohol and embed slides

Besides visual inspection of sections under the microscope, amyloidstaining was additionally quantified by optically excising 10 randomlyselected plaques from the histological images using the ImagePro 5.0image analysis system and determining their average greyscale value.Optical density values (were calculated from the greyscale values bysubtracting the mean background density of the stained material from thedensity of amyloid plaques (0%—no plaque staining above surroundingbackground, 100%—no transmission/maximal staining). The differencesbetween between antibodies 6E10/4G8 and 6G1, 8C5 and 8F5, respectively,were tested for statistical significance with ANOVA.

Results:

All antibody-stained material described proved to be congophilic amyloiddeposits (FIG. 7(A)). The globulomer-preferring antibodies 8F5 and 8C5stained parenchymal and meningeal congophilic deposits of Aβ peptidesignificantly less than the antibodies 6G1 and 6E10 (FIG. 7(B)-(C),(H)).Quantitative analysis of parenchymal amyloid plaque staining revealedbinding of all antibodies to plaques (statistically significant densityabove control), but binding of antibody 8F5 and 8C5 was significantlylower than binding of the reference antibody 6E10 (raised to N-terminalsequence of Aβ) and equal or lower than reference antibody 4G8 (raisedto N-terminal sequence of Aβ) (FIG. 7(D)-FIG. (G)), Antibodies 8F5 and8C5 bind less to amyloid deposits than antibodies which recognize Aβmonomer or part of the Aβ sequence. Treatment with antibodies binding tofibrillar Aβ peptide can lead to fast dissolution of amyloid plaques inbrain tissue and a subsequent increase of soluble Aβ concentration,which in turn is thought to be neurotoxic and could lead tomicrohemorrhages, and/or a fast dissolution of vascular amyloid, whichalso could lead to microhemorrhages. Therefore, an antibody therapy thataffects the soluble globulomer rather than the monomer is preferred.

1-56. (canceled)
 57. A method of treating or preventing Alzheimer'sDisease in a patient in need of said treatment or prevention, the methodcomprising the steps of: administering to a patient an isolated antibodyin an amount sufficient to effect said treatment or prevention in thepatient, wherein the antibody is a monoclonal antibody comprising: i) aCDR 1 of the light chain variable region of the monoclonal antibodycomprising the amino acid sequence of SEQ ID NO:8; ii) a CDR 2 of thelight chain variable region of the monoclonal antibody comprising theamino acid sequence of SEQ ID NO:9; iii) a CDR 3 of the light chainvariable region of the monoclonal antibody comprising the amino acidsequence of SEQ ID NO:10; iv) a CDR 1 of the heavy chain variable regionof the monoclonal antibody comprising the amino acid sequence of SEQ IDNO:5; v) a CDR 2 of the heavy chain variable region of the monoclonalantibody comprising the amino acid sequence of SEQ ID NO:6; and vi) aCDR 3 of the heavy chain variable region of the monoclonal antibodycomprising theamino acid sequence of SEQ ID NO:
 7. 58. A method oftreating or preventing Alzheimer's Disease in a patient in need of saidtreatment or prevention, the method comprising the steps of:administering to a patient an isolated antibody in an amount sufficientto effect said treatment or prevention in the patient, wherein theantibody is a monoclonal antibody comprising the complementaritydetermining regions of SEQ ID NO:3 and SEQ ID NO:4, wherein saidantibody is murine, human or humanized.
 59. A method of treating orpreventing Alzheimer's Disease in a patient in need of said treatment orprevention, the method comprising the steps of: administering to apatient an isolated antibody in an amount sufficient to effect saidtreatment or prevention in the patient, wherein the antibody is amonoclonal antibody which specifically binds to forms of amyloid beta,wherein: i) the light chain variable region of the monoclonal antibodycomprises the amino acid sequence of RSSQSLVYSNGDTYLH (SEQ ID NO: 8);the amino acid sequence of KVSNRFS (SEQ ID NO: 9); and the amino acidsequence of SQSTHVPWT (SEQ ID NO: 10); and ii) the heavy chain variableregion of the monoclonal antibody comprises the amino acid sequence ofGFTFSSYGMS (SEQ ID NO: 24); the amino acid sequence of SINSNGGSTYYPDSVKG(SEQ ID NO: 6); and the amino acid sequence of GDY (SEQ ID NO: 25). 60.A method of treating or preventing Alzheimer's Disease in a patient inneed of said treatment or prevention, the method comprising the stepsof: administering to a patient an isolated antibody in an amountsufficient to effect said treatment or prevention in the patient,wherein the antibody is a monoclonal antibody comprising: i) a CDR 1 ofthe light chain variable region comprising the amino acid sequence ofSEQ ID NO:8; ii) a CDR 2 of the light chain variable region comprisingthe amino acid sequence of SEQ ID NO:9; and iii) a CDR 3 of the lightchain variable region comprising the amino acid sequence of SEQ IDNO:10; and iv) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:3.
 61. A method of treating or preventingAlzheimer's Disease in a patient in need of said treatment orprevention, the method comprising the steps of: administering to apatient an isolated antibody in an amount sufficient to effect saidtreatment or prevention in the patient, wherein the antibody is amonoclonal antibody comprising: i) a CDR 1 of the heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:5; ii) a CDR 2 ofthe heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:6; iii) a CDR 3 of the heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:7; and iv) a light chain variableregion comprising the amino acid sequence of SEQ ID NO:4.
 62. The methodof claim 57, 58, 59, 60 or 61 wherein said isolated antibody isadministered via a route selected from the group consisting ofintramuscular administration, intravenous administration andsubcutaneous administration.
 63. A method of preventing or treatingAlzheimer's Disease in a patient in need of said prevention or treatmentcomprising the step of administering a vaccine to a patient in an amountsufficient to effect said prevention or treatment, wherein the vaccinecomprises an antibody, wherein the antibody is a monoclonal antibodycomprising: i) a CDR 1 of the light chain variable region of themonoclonal antibody comprising the amino acid sequence of SEQ ID NO:8;ii) a CDR 2 of the light chain variable region of the monoclonalantibody comprising the amino acid sequence of SEQ ID NO:9; iii) a CDR 3of the light chain variable region of the monoclonal antibody comprisingthe amino acid sequence of SEQ ID NO:10; iv) a CDR 1 of the heavy chainvariable region of the monoclonal antibody comprising the amino acidsequence of SEQ ID NO:5; v) a CDR 2 of the heavy chain variable regionof the monoclonal antibody comprising the amino acid sequence of SEQ IDNO:6; and vi) a CDR 3 of the heavy chain variable region of themonoclonal antibody comprising the amino acid sequence of SEQ ID NO: 7.64. A method of preventing or treating Alzheimer's Disease in a patientin need of said prevention or treatment comprising the step ofadministering a vaccine to a patient in an amount sufficient to effectsaid prevention or treatment, wherein the vaccine comprises an antibody,wherein the antibody is a monoclonal antibody comprising thecomplementarity determining regions of SEQ ID NO:3 and SEQ ID NO:4,wherein said antibody is murine, human or humanized.
 65. A method ofpreventing or treating Alzheimer's Disease in a patient in need of saidprevention or treatment comprising the step of administering a vaccineto a patient in an amount sufficient to effect said prevention ortreatment, wherein the vaccine comprises an antibody, wherein theantibody is a monoclonal antibody which specifically binds to forms ofamyloid beta, wherein: i) the light chain variable region of themonoclonal antibody comprises the amino acid sequence ofRSSQSLVYSNGDTYLH (SEQ ID NO: 8); the amino acid sequence of KVSNRFS (SEQID NO: 9); and the amino acid sequence of SQSTHVPWT (SEQ ID NO: 10); andii) the heavy chain variable region of the monoclonal antibody comprisesthe amino acid sequence of GFTFSSYGMS (SEQ ID NO: 24); the amino acidsequence of SINSNGGSTYYPDSVKG (SEQ ID NO: 6); and the amino acidsequence of GDY (SEQ ID NO: 25).
 66. A method of preventing or treatingAlzheimer's Disease in a patient in need of said prevention or treatmentcomprising the step of administering a vaccine to a patient in an amountsufficient to effect said prevention or treatment, wherein the vaccinecomprises an antibody, wherein the antibody is a monoclonal antibodycomprising: i) a CDR 1 of the light chain variable region comprising theamino acid sequence of SEQ ID NO:8; ii) a CDR 2 of the light chainvariable region comprising the amino acid sequence of SEQ ID NO:9; andiii) a CDR 3 of the light chain variable region comprising the aminoacid sequence of SEQ ID NO:10; and iv) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:3.
 67. A method ofpreventing or treating Alzheimer's Disease in a patient in need of saidprevention or treatment comprising the step of administering a vaccineto a patient in an amount sufficient to effect said prevention ortreatment, wherein the vaccine comprises an antibody, wherein theantibody is a monoclonal antibody comprising: i) a CDR 1 of the heavychain variable region comprising the amino acid sequence of SEQ ID NO:5;ii) a CDR 2 of the heavy chain variable region comprising the amino acidsequence of SEQ ID NO:6; iii) a CDR 3 of the heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:7; and iv) a light chainvariable region comprising the amino acid sequence of SEQ ID NO:4.